Non-destructive orientation mapping is an important characterization tool in materials science and geoscience for understanding and/or improving material properties based on their grain structure. Confocal Raman microscopy is a powerful non-destructive technique for chemical mapping of organic and inorganic materials. Here we demonstrate orientation mapping by means of Polarized Raman Microscopy (PRM). While the concept that PRM is sensitive to orientation changes is known, to our knowledge, an actual quantitative orientation mapping has never been presented before. Using a concept of ambiguity-free orientation determination analysis, we present fast and quantitative single-acquisition Raman-based orientation mapping by simultaneous registration of multiple Raman scattering spectra obtained at different polarizations. We demonstrate applications of this approach for two- and three-dimensional orientation mapping of a multigrain semiconductor, a pharmaceutical tablet formulation and a polycrystalline sapphire sample. This technique can potentially move traditional X-ray and electron diffraction type experiments into conventional optical laboratories. Although polarized Raman microscopy is sensitive to orientation changes, quantitative information has been missing. Here, the authors use simultaneous registration of multiple Raman scattering spectra obtained at different polarizations and show quantitative orientation mapping Many materials are polycrystalline, meaning that they are composed of a large number of grains  of different crystallographic orientations. A full three-dimensional (3D) orientation mapping of the grains  is crucial to relate structure to properties.2, but as a surface-based method, it can only be extended to 3D by serial sectioning. Moreover, it has challenges in the analysis of semiconductors and dielectric materials1. Transmission electron microscopy can provide 3D orientation maps with 1 nm resolution, but only in thin foils3. On the other hand, imaging based on X-ray diffraction using synchrotrons can provide 3D maps of thousands of grains in mm-sized samples with a resolution of 2 µm5—in scanning6 or microscopy mode7 even down to 100 nm—but this is an expensive and infrastructure-demanding technique. Recently developed mapping of grain orientations in 3D by laboratory X-rays (LabDCT) is a good alternative to the synchrotron measurements; however, its spatial resolution is limited to 10–15 µm8.Electron backscattering diffraction (EBSD) is a widespread technique for such orientation mapping9, with a diffraction-limited spatial resolution of around 200 nm10. In PRM, if the symmetry of the crystal is known, Raman tensors j can be obtained11 and Raman intensities I(θ) versus sample rotation angle θ can be simulated9. Correlation between theoretical and experimental angular intensity dependencies Itheor(θ) and Iexp(θ) can, therefore, be used for the determination of the local crystallographic orientation9  has the potential to be developed into a relatively cheap orientation mapping technique41. There has been a growing interest in confocal PRM studies of polycrystalline ceramics during the last years43 including piezoelectric ceramics46. More recently an orientation type contrast was exploited in PRM studies of uranium oxide ceramics47 and cancerous breast tissue48. In these papers, the PRM setup was used for improving the contrast in crystal features differentiation. The 3D domain orientation in BaTiO3 crystals has been revealed after the sample has been mapped in a polarized Raman microscope with an a-plane crystal orientation49. Orientation distribution mapping of polycrystalline CuInSe2 has also been demonstrated10 by a combination of Raman contrast and EBSD mapping.Most of the listed papers were focused on the determination of sample crystallographic orientations at the microscale, with the Raman microscope working in backscattering mode9. Such optical layouts require multiple Raman spectra measurements using numerous combinations of the orientation of the polarization state of the incident laser and orientation of the analyzer41. Due to this, Raman orientation mapping would need sequential polarization measurements at each raster-scanning point, which would make such hypothetical mapping procedures very complex and extremely slow and thus practically impossible. In addition, existing experimental PRM setups introduce ambiguities in measurements, implying that the approach fails completely for some orientations. As an example, in a scheme with normal sample illumination and on-axis collection of scattered light, it is impossible to determine a rotation of a (111) Si wafer around the [111] axis  and is well investigated by PRM11   wafer in misorientation plots in Supplementary Fig. Orientation fitting was done using varying numbers of polarized channels (2–12 channels), see Supplementary Note Simulation results shown in Supplementary Note ψ which differs by 180°. Adding off-axis measurement channels removes this ψ + 180° ambiguity.To assess the quality of the orientation determination in the general case, additional analysis using exhaustive search of indistinguishable solutions over a range of all possible Euler angles was performed  from the three incident lasers is divided into nine backscattering channels (six on-axis and three off-axis) after propagation through three analyzers with orientations 0° and 90° for on-axis Raman scattering detection and 90° for off-axis Raman scattering detection. The information obtained from the nine Raman channels is used for determining the crystallographic orientation of a selected local volume. 2D or 3D orientation mapping is obtained by scanning the sample in x, y, z with respect to the incident beams.The chosen geometry with nine polarized channels is illustrated in Fig. Based on theoretical investigation of quantitative orientation mapping by Raman microscopy, we have developed and demonstrated a method, Single-Acquisition Raman Orientation Mapping (SAROM), for the study of crystalline samples. Our self-made Raman setup provides two orders of magnitude faster Raman polarization measurements compared with existing techniques and the ambiguity issue is overcome. The SAROM system is capable of simultaneously illuminating the sample with multiple laser beams at different orientations of the laser polarization state and detecting Raman scattering beams at multiple on-axis/off-axis scattering directions without using any moving parts Fig. . As showSAROM consists of a Laser Beam Delivery System (LBDS), which directs three laser beams with different orientations of polarization state on the sample focal plane Fig. , and an 53, Raman intensity scaling and normalization43, Linear Phase (LP) and Linear Amplitude (LA) anisotropy of each optical element54, and depth-dependent birefringence13. Since each channel has its own correction parameters, an experimentally based calibration becomes practically impossible. Therefore, we build a model, which predicts the values of the listed parameters  of the microscope objective in PRM, has been realized by taking into account the full aperture of the microscope objectiveIn the following, we demonstrate the method with a few examples. As an application in 2D, we present work on a polycrystalline Si (poly-Si) solar cell. The SAROM scanning procedure used for the poly-Si sample is shown in Supplementary Movie 55, SAROM can be used for the study of extended defects like dislocations and grain boundaries, which e.g., directly correlate with the efficiency of solar cells.Similar to EBSD56. It has been shown that PRM can be used to visualize particles on the surface of tablet formulations57, however so far no quantitative information on the orientation of single particles has been provided. Here, we performed a content uniformity Raman measurement on a tablet containing carbamazepine dihydrate (CBZD) and polyvinylpyrrolidone (PVP). Using MCR, these components were decomposed including the fluorescence background as shown in Fig. 2h crystal class)  mode are shown in Supplementary Figs. Following the SAROM workflow  Raman mode, corresponding to the nine differently polarized Raman channels, are shown in Supplementary Fig. 59.Nine maps of the intensity of Eg Raman moSAROM has some limitations. The theoretically determined orientation resolution varies with the orientation of the grain. Typically, the theoretical variation is well below 1° , ceramics , and superhard materials . Moreover, the functionality of SAROM can be extended by combination with e.g., confocal Raman microscopy, hyperspectral imaging, or polarized light microscopy. Further development of SAROM can be expanded into instantaneous polarized Raman mapping similar to wide-field Raman microscopy68.In summary, SAROM is a nondestructive quantitative orientation mapping method with a high diffraction-limited spatial resolution, similar to that of confocal Raman microscopy , (110), (111) were provided by National Center of Micro- and Nanofabrication in Denmark.Polycrystalline Si solar cell was provided by the factory Pillar .Anhydrous carbamazepine was obtained from Sigma-Aldrich (CAS No.298-46-4). Povidone  was obtained from Sigma-Aldrich (CAS No. 9003-39-8).Sapphire monocrystalline plates at a-, c-, m-, and r-planes were purchased from Crystran Ltd.Polycrystalline sapphire sample with dimensions 10 × 5 × 5 mm and average grain size of 20 µm was purchased from CoorsTek GmbH.The surface of the polycrystalline Si was mechanically polished prior to EBSD investigation. Silica microparticles having dimensions of 6, 3, and 1 µm were sprayed on a rotating diamond grinding disc for the polishing. Starting with the larger microparticle size, the sample was alternately polished and visually inspected using an optical microscope. The inspection was necessary to verify that the surface was without scratches along a particular direction, index of an inhomogeneous polishing. The same polishing procedure was repeated using each microparticle size.The polished sample was mounted on an EBSD holder with a 54° tilt respect to the electron beam perpendicular. The holder was installed on a FEI Nova 600 NanoSEM stage and the sample was tilted 16°, to reach a total tilt of 70° respect to the electron beam perpendicular. The microscope was equipped with a Bruker QUANTAX EBSD detector and operated at an accelerating voltage of 15 kV, a beam current of 10 nA, a 40 µm aperture and at a working distance of 12 mm. The detector was positioned in such a way that the smallest distance between the electron-beam focusing point at the specimen surface and the camera was 16.5 mm. All measurements were performed in high vacuum mode. EBSD orientation map was collected with a pattern resolution of 320 × 240 pixels, exposure time of 155 ms, and step size of 25 µm, total measurement time 3.3 h. The raw data were processed using MTEX toolbox in Matlab.Anhydrous carbamazepine was dissolved (25 mg/mL) in a vial filled with a water–ethanol solvent composition (3:1) at 60 °C. The vial was then removed from the hot plate and allowed to cool down to room temperature. After around 4 days, needle and plate shaped particles of CBZD formed via slow evaporation crystallization and were harvested by filtration.λ = 1.5418 Å) source that was generated at a tube voltage of 45 kV and current of 40 mA. A PIXcel detector with a θ/θ goniometer was used. The measurements were performed at a reflection mode between 5° and 35° at a step size of 0.0263° 2θ and a scan speed of 0.06734° 2θ. The X’Pert Data Collector software provided by PANalytical, Almelo, Netherlands was used in order to analyze the diffractograms. For reproducibility purposes, triplicate measurements were performed.CBZD was identified using a PANalytical Xpert PRO X-Ray Diffractometer  with a nickel filtered CuKα , intermolecular interactions, and different faces (SF3) of CBZD, Mercury CSD 3.10.3 (Build 205818) was used. Mercury was provided by the Cambridge Crystallographic Data Centre, UK.A tablet press  with a 6 mm diameter cylindrical flat-faced compacts was used in order to prepare the tablets. This tablet press was equipped with a 500 kg load cell 8CT6–500–022). 200 mg (±10 mg) of CBZD and 100 mg (±10 mg) were compacted at a compression speed of 60 mm/min. The tableting die was lubricated with magnesium stearate suspension prepared in acetone. Tablets were prepared in triplicate.69 from the Cambridge Structural Database (CSD).X-ray powder diffraction (XRPD) was used in order to confirm carbamazepine (CBZ) to be carbamazepine dihydrate (CBZD). In order to achieve this, the experimentally obtained XRPD diffractogram of CBZD was compared with the calculated diffractogram In order to obtain high-quality 2D/3D Raman orientation maps, we developed and experimentally verified all required data acquisition and data analysis steps. The overview of SAROM workflow is shown in Supplementary Fig. 41 in order to find the minimum number of polarization measurements required for obtaining valid crystal orientations One of the most critical steps which needed to be solved in SAROM is achievement of high speed of polarization measurements. For this purpose, we designed and constructed a Raman setup without moving parts, capable to illuminate the sample with multiple laser beams at different laser polarizations and simultaneously detect Raman scattering beams at multiple analyzer orientations , (110), and (111), respectively. It was determined that at least three differently polarized laser orientations and three analyzer orientations are required, with mandatory presence of off-axis channels. In total, we got nine combinations of polarization measurements which can be defined as 00, (110)entclass1pt{minima1, L2, L3 operated at slightly different wavelengths 781 nm, 783.5 nm and 784.8 nm, respectively. Wavelength of each laser can be finely adjusted by the change of diode temperature in order to get the best spectral distance between vibrational modes. The laser beams have polarization angles x axis in laboratory coordinate system  , which provides simultaneous measurements at three different analyzer orientations: 0° and 90° in on-axis registration and 90° in off-axis registration.The key module of RBDS is the WAU. It consists of quartz Wollaston prism, analyzer, and mask   were used.The laser clean-up filters Lf1-Lf4  are placed on the beam path after the laser output in order to block unwanted emission background from the laser pumping, reducing its intensity of about six orders of magnitude.In 2D-SAROM setup, the collimated beam from laser was expanded in beam expander  and f2 = 100 mm ) up to the diameter of 10 mm. Expanded beam was divided into three beams by a set of beamsplitters BS1-BS3 and polarized BS1 .In 3D-SAROM setup, we used three diode lasers stabilized at different temperatures in order to control laser wavelength. Each laser beam propagates through separate beam expanders . Final beam diameter for three laser beams was 10 mm. All beams combined by polarized beamsplitter BS2  and non-polarized beamsplitter BS4 .The collimated beams from LBDS in 2D and 3D configurations reflect from mirrors M1-M6  and pass through a motorized power attenuation filter wheel  with seven optical density (OD) filters inside.SAROM can be commutated to the 2D-SAROM or 3D-SAROM geometries by switching of the motorized flipper mirror MFM .Then the laser beams reflect from dichroic mirror DM1  and passes through the dichroic mirror DM2 responsible for the coupling with a visible microscope. Finally, laser beams are delivered to the custom made microscope objective  and imaging CCD . White light from the LED collimates and passes through the edge filter , in order to block the NIR part of the LED emission spectrum. In such a way a visible real-time image can be monitored during the registration of Raman spectra . Lens f3 is a NIR achromatic doublet pair which focuses Raman beam on the motorized spectrograph slit .Spectrograph consists of NIR achromatic doublet pair lenses f4 , custom designed Double-Gauss focusing lens f5, transmission fused silica polarization independent grating (Light Smith) and edge filter Ef2 . Raman spectra collected on the air cooled CCD .−1 from laser line was at the level of 2.1 × 10−4 which is very good result taking into account relatively small spectrograph dimensions and focal lengths. Spectrograph inside view is shown in Supplementary Fig. Aberration corrected design of the spectrograph and double-gauss lens design are shown in Supplementary Figs. In the case of SAROM microscopy of mono- and polycrystalline Si laser power on the sample in each polarized channel was 50 mW, laser spot size was 3 µm, exposure time per mapping point was 0.05 s, CCD readout mode was multitrack.In the case of SAROM microscopy of CBZD drug laser power on the sample in each polarized channel was 10 mW, laser spot size was 3 µm, exposure time per mapping point was 0.15 s, CCD readout mode was multitrack.In the case of SAROM microscopy of mono- and polycrystalline sapphire laser power on the sample in each polarized channel was 100 mW, laser spot size was 0.8 µm, exposure time per mapping point was varied from 50 ms to 2 s , CCD readout mode was multitrack.Supplementary InformationArticle File - Peer Review FileDescription of additional supplementary filesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5


DamID, a method to identify DNA associating with a particular protein, was originally developed for use in immortalized tissue culture lines. The power of this technique has led to its adaptation for a number of additional systems. Here we report adaptations for its use in primary cells isolated from rodents with emphasis on the challenges this presents. Specifically, we present several modifications that allow the method to be performed in mouse acutely isolated primary hepatocytes while seemingly maintaining tissue genome architecture. We also describe the downstream bioinformatic analysis necessary to identify LADs and discuss some of the parameters and their effects with regards to the sensitivity of the method. Following in situ labeling, genomic DNA is extracted from cells and then the uniquely methylated GATC-motifs are specifically cut by the DpnI restriction enzyme. Subsequent digestion with DpnII, which only cuts at unmethylated GATC sequences, facilitates the selective amplification of genomic fragments that are flanked by methylated GATC motifs after ligation of adaptors to the DpnI-exposed ends. Because a wide range of fragment sizes would be expected from the genome, the resulting DNA appears as a smear on agarose gels with no clearly delineated individual bands. The amplified fragments were originally identified using whole genome microarrays, but are now more commonly identified by sequencing due its simplicity and the wide range of organisms for which the procedure is now used that include mammalian cells Schizosaccharomyces pombe[14], Caenorhabditis elegans[15], Drosophila melanogasterArabidopsis thalianaThe original LaminB1-DamID gets around this by fusing the insoluble nuclear Lamin B1 protein with a bacterial Dam methylase. The fusion protein localizes to the nuclear envelope while the Dam methylase recognizes GATC motifs in the DNA in its proximity and catalyzes the addition of a methyl group to the adenine to form m6A. This kind of DNA methylation is unique to prokaryotes, and permits the identification of DNA in close proximity with the nuclear envelope in higher organisms. The use of LaminB1 is advantageous for the higher eukaryotes that express them because this protein is exclusively at the nuclear envelope due to its permanent farnesylation Drosophila GATC sites within 2.5–5 Kb of a locus where the Dam methylase is bound have been reported to be efficiently methylated DpnI fragment size of around 260 bp, and 99.9% under 3 Kb.A major advantage of DamID over ChIP is that DamID does not rely on the availability of antibodies and avoids issues related to epitope masking Previously, we modified the DamID procedure when we used it to follow gene positioning changes during myogenesis because we found that in muscle cells there was a very high background of mitochondrial genomes carrying similar methylation, which needed to be overcome 22.1ad libitum. All the experimental procedures were carried out in accordance with European Directive (2010/63/EU) and approved by the Institutional Animal Care and Use Committee of Perugia University and the Italian Ministry of Health. Primary hepatocytes were isolated from12 to 16-week old male mice by a two-step collagenase perfusion method, as previously reported 2+- and Mg2+-free buffer  at a rate of 5 ml/min for 5 min at 37 °C . Next, collagenase solution , 15 mM HEPES, 100 U/ml penicillin, and 0.1 mg/ml streptomycin), equilibrated at 37 °C, was perfused to digest the liver . After digestion, gall bladder was removed and the liver was placed in a Petri dish with 10 ml of warm (37 °C) collagenase solution. Hepatocytes were released from the liver capsule by tearing liver lobes with two pairs of forceps and gently shaking the tissue under a tissue culture hood to obtain a cloudy cell suspension that then was filtered through a 70 µm nylon cell strainer . Intact hepatocytes were washed with complete cell culture medium , 2 mM l-glutamine, 0.1 µM insulin, 0.1 µM dexamethasone, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 40 µg/ml gentamicin) by three rounds of centrifugation at 50×g for 2 min at 4 °C. Cells were resuspended in complete culture medium and cell count was performed by trypan blue exclusion assay. Only preparations with a viability exceeding 85%, were used for the experiments. Cells were seeded at 60–70% of confluence into dishes precoated with collagen from rat tail (5 µg/cm2) in complete culture medium. Cells were allowed to attach for 2 h, gently washed once with PBS, then fresh medium was added for additional 2 h. Thereafter, cells were grown in complete serum-free culture medium. The primary hepatocytes were cultured at 37 °C in a humidified 5% CO2 incubator with daily medium renewal. Immediately after isolation, hepatocytes were tested for mycoplasma infection by a PCR based method using genomic DNA as template (20–40 ng per reaction), as previously described The specific procedure described here used C57BL/6 mice, though it should be generally applicable to different mouse strains and other mammals. The animals were maintained at a constant temperature of 22 °C, 12 h light/dark cycle, and given access to food and water 2.2DamID relies on the efficient transfer of a transgene expressing low levels of the protein of interest fused to a bacterial Dam methylase For each experimental condition three lentiviruses are required: 1) protein of interest, in our case LaminB1, fused to the bacterial Dam methylase, 2) Dam methylase alone, and 3) empty vector, a technical control. Comparing the signal levels from Dam-LaminB1 against soluble Dam alone controls for differences in accessibility to chromatin and sequence-dependent biases during subsequent PCR amplification. The empty vector control is very important, as in mammalian cells there should not be any other source of m6A than the DNA methylated by the exogenously introduced Dam-encoding constructs. The presence of amplified DNA in the empty vector control is often an indication of mycoplasma contamination or some other source of prokaryotic infection. If this happens, prokaryotic DNA would comprise a very high proportion of the recovered methylated DNA, severely compromising the experiment.2.2.12using high glucose DMEM supplemented with 10% fetal bovine serum, 6 mM l-Glutamine, 50 U/ml penicillin, 0.05 mg/ml streptomycin, and 0.5 mg/ml Geneticin (to maintain expression of large T-antigen). Cells were split by trypsinization 1:4–1:6 when they reached 80–90% of confluence. It is important to avoid letting the cells get too confluent and to ensure the cells are actively dividing to maximize transfection efficiency.HEK293FT cells (Clontech) were grown at 37 °C and 5% CO2.2.2Non-replicative self-inactivating lentiviruses were generated by cotransfection of psPAX2 , pMD2.G (VSV-G envelope protein) and transfer vectors encoding either a bacterial Dam methylase fused to LaminB1 (pLgw Dam-LaminB1), Dam methylase alone (pLgw Dam) or an empty vector (pLgw-empty) as a control. Lentiviral plasmids and the DamID backbone vectors are available at Addgene. Transfer vectors used here were a gift from Bas van Steensel. This is a 2nd generation lentiviral system is sufficiently safe for most purposes. It's worth noting, however, that there are other systems (3rd and 4th generation) that confer increased levels of safety. The drawback is that they typically yield lower viral titres because they require the co-transfection of a larger number of plasmids. The user should consider the nature of the gene of interest and consult their local regulations before deciding whether a 2nd generation system or a safer alternative should be employed.6 cells per dish, in 5 ml of culture medium. It is not necessary to omit antibiotics during transfection, but it may help to obtain higher yields. After incubation, the lipofection mix was added to the cells, still in suspension, mixed gently, and the dishes placed back into the incubator overnight.For each transfection we prepared one 1.5 ml microfuge tube containing 4.6 µg of psPAX2, 2.8 µg of pMD2.G and 7.5 µg of transfer vector in 1mlOpti-MEM, and one 15 ml tube with 36 µl of Lipofectamine 2000 in 2 mlOpti-MEM. Following a 5 min incubation at room temperature, the DNA solution was added to the Lipofectamine, vortexed briefly, and incubated at room temperature for 15–20 min. Meanwhile, HEK293FT cells were harvested, counted with a hemocytometer, and seeded in 10 cm dishes at 6 × 102.2.3g to remove cellular debris. The supernatant was carefully aspirated into fresh tubes, LentiX Concentrator (Clontech) added in a 3:1 ratio, mixed gently, and incubated overnight at 4 °C. Lentivirus particles were then precipitated by centrifugation at 1500×g for 45 min, and resuspended in 200–400 µl Opti-MEM. The lentiviral solutions were divided into 20–50 µl aliquots and stored at −80 °C. It is important to avoid freeze-thaw cycles to ensure the viability of the virus. Alternatively, instead of using polyethylene glycol (PEG) based reagents such as LentiX that allow the use of benchtop centrifuges most commonly found in tissue culture labs, it is possible to precipitate the lentivirus by ultracentrifugation, as described Transfection medium was removed after 16–18 h and 12–14 ml of fresh culture medium added to each 10 cm dish. The supernatant was harvested 48 h later and centrifuged for 15 min at 500×2.3To perform lentiviral transduction of primary mouse hepatocytes under optimal conditions for successful application of the DamID protocol, cells are initially tested for tolerance to polybrene, a transduction enhancer, and for the reporter gene expression levels . Polybrene favors lentiviral particles association with target cell plasma membranes by reducing negative charge repulsion forces. However, excessive amounts can lead to apoptosis, so it is important to test for toxicity first and use a concentration that does not cause harm to cells (at least in the relatively short period of the infection), and that it does not interfere with the experiment in some other way. There are other transduction enhancers that can be used, such as protamine sulphate, retronectin, DEAE-dextran, and various proprietary commercial others. In fact, we preferred protamine sulphate in the case of mouse myogenic differentiation system because polybrene appeared to interfere with differentiation 2.3.16 cells/well) and after 4 h they were incubated with serum-free culture medium in the presence or in the absence of epidermal growth factor  and increasing polybrene concentrations (0–10 µg/ml). The medium was replaced daily by fresh medium supplemented with EGF and polybrene. Cell morphology changes were periodically monitored by direct observation with a light microscope; images were acquired by a phase-contrast microscope equipped with a digital camera. After 72 h of incubation in the presence of polybrene, cell viability was assessed by using a modification of the 3--2,5-diphenyl tetrazolium bromide (MTT) reduction assay, which has been applied to assess cytotoxicity in hepatocytes 6 cells per well and, after the treatments, they were detached by trypsinization. Recovered cells were resuspended in 0.5 ml of PBS containing 0.5 mg/ml of MTT and incubated for 1 h at 37 °C with gentle shaking. After centrifugation (5 min at 300×g), cell pellets were dissolved in 1 ml of dimethyl sulfoxide (DMSO) by pipetting and cellular debris were removed by further centrifugation2 min at 13000×g. Absorbance of the reduced MTT product (formazan) was measured at 595 nm by a UV–VIS spectrophotometer .Freshly isolated hepatocytes were seeded into 24-well plates (0.1 × 102.3.26 cells/well) and cultured in 0.8 ml of transduction medium per well with the addition of different amounts of lentiviral preparation . Whole cell protein lysates were obtained at 72 h after transduction by the direct addition of 150 µl per well of boiling lysis buffer . After protein quantitation by Bradford assay, samples were adjusted to 2% SDS, 1.5% β-mercaptoethanol, and 0.05% bromophenol blue, subjected to SDS-PAGE (30 µg of protein per lane), and transferred to nitrocellulose or PVDF membranes. Filters were probed overnight with anti-GFP  or anti-β-tubulin  primary antibodies and afterwards with a HRP-conjugated goat anti-mouse secondary antibody . Proteins of interest were visualized using ECL reaction and images were acquired by a ChemiDoc™ imaging system. To directly visualize EGFP-derived fluorescence in transgene-expressing cells, hepatocytes were grown on 12 mm coverslips (Knittel Glass) inserted into 24-well plates (0.1 × 106 cells/well) and transduced for 72 h as described above, with 1/8 of starting medium volume of the lentiviral preparation encoding for the EGFP. After the incubation period, cells were fixed with 4% PFA and quenched in 50 mM ammonium chloride for 15 min at room temperature To test the efficiency of transgene expression in isolated mouse hepatocytes, attached cells were grown for 18 h in culture medium enriched with EGF (40 ng/ml), polybrene (6 µg/ml), and EGFP-encoding lentiviral particles previously made using pRRLSIN.cPPT.PGK-GFP.WPRE (Addgene). Transduction medium was then removed, hepatocytes were washed with PBS, and wells were replenished with fresh culture medium containing EGF on a daily basis. For the analysis of exogenously expressed protein by immunoblotting, cells were seeded on 6-well plates  and empty, Dam alone-, orDam-LaminB1-encoding lentiviruses (transduction medium). The transduction medium was then replaced with fresh serum-free complete medium with EGF and transduced hepatocytes were left in the incubator for a further 48 h to ensure sufficient Dam-fusion protein expression and, therefore, methylation of the DNA sequences interacting with Lamin B1.Cells were isolated as outlined above, seeded into 6-well plates at a density of 0.5 × 102.4.2g for 5 min and resuspended in 0.2 ml of PBS. RNAseA (0.1 mg/ml) treatment was performed for 3 min prior the addition of cell lysis buffer. Complete cellular digestion was obtained through the incubation with proteinase K (30 min at 56 °C). Digested samples were loaded onto spin columns for genomic DNA purification. Elution of genomic DNA from the columns was performed twice with 100 µl of buffer AE (preheated at 65 °C) followed by incubation (1 min at room temperature) and centrifugation at 6000×g for 1 min. Next, DNA was precipitated by the addition of 0.1 volumes of 3 M sodium acetate (pH 5.4), 10 µg of glycogen, and 3 volumes of 100% ethanol. Samples were incubated at −80 °C for 30 min and DNA was pelleted at 13000×g for 15 min at 4 °C. DNA pellets were washed with 400 µl of 70% ethanol by centrifugation at 13000×g for 5 min, at 4 °C. Air-dried DNA was resuspended in 13 µl of TE-lowE  buffer at 56 °C for 1 h. DNA concentration was determined by a microplate reader  equipped with a NanoQuant plate.After 72 h of transduction, genomic DNA was extracted by using the DNeasy blood and tissue kit  according to manufacturer’s instructions. Briefly, cells were trypsinized, centrifuged at 300×2.4.3DpnI in a total volume of 10 µl using restriction buffer 4 from NEB. DpnI only cuts GATC sequences when the adenine is methylated. An additional 2.5 µg DNA aliquot should be set in a parallel reaction lacking DpnI, as a control. This control should not generate any significant amount of amplification. If it does, it would suggest widespread DNA fragmentation has occurred, such as apoptosis-induced DNA cleavage and the samples should not be used. Note that a pretreatment with alkaline phosphatase could be used to prevent ligation of the DamID adaptors to the damaged DNA, and thus its amplification, however we recommend this only as a last resort if a new sample cannot be easily obtained, because a large amount of apoptotic cells in the sample could affect the resulting DamID profile. After digestion, DpnI is inactivated by heating to 80 °C for 25 min, and then the dsAdR adaptor  is ligated to the DpnI digested DNA. This is achieved by adding 2 µl of 10× T4 ligase buffer (NEB), 5 units of T4 DNA ligase (NEB), dsAdR adaptor to 2 µM, and nuclease-free water to a final volume of 20 µl, and incubating at 16 °C for 16 h. Omit the T4 ligase on one of the duplicate DpnI positive reactions. This control is to ensure that amplification is not due to dsAdR-independent PCR of similar sequences that might be present in the genome. If the yield of DNA is low and there is not enough for all the controls, this one is probably the safest one to omit. Ligase can be inactivated by heating at 65 °C for 20 min. Unmethylated GATC sequences are then digested using DpnII (which cuts only unmethylated GATC) by adding 5 µl of 10xDpnII buffer (NEB), 10 units of DpnII (NEB), and nuclease-free water to make up a total of 50 µl, and incubated at 37 °C for 1 h.DNA methylated by the exogenous Dam constructs can be specifically amplified by PCR, after processing it to obtain DNA fragments flanked by methylated GATC sequences to which adaptors have been ligated. The whole process has been designed to be performed using a single PCR tube to minimize losses. For each experiment 3 aliquots of 2.5 µg are required. First, two aliquots of 2.5 µg of genomic DNA are digested overnight at 37 °C with 10 units of At this stage, DNA fragments that were flanked by methylated DNA can be enriched by PCR, as they should have a dsAdR adaptor at both ends. Transfer 5 µl of the products (about 250 ng DNA) to a fresh PCR tube, and add 1.25 µl of 50 µM AdR-PCR primer (GGTCGCGGCCGAGGATC), 1 µl of 10 mM dNTP mix, 5 µl of 10× PCR buffer and 1 µl of Advantage PCR enzyme mix (Clontech 639105), and nuclease free water to 50 µl. We have tried other PCR reagents but this specific kit is superior to anything else we have tried. Run the following program: [10 min @ 68 °C] × 1,  × 1,  × 4,  × 18. Run 3 µl of each reaction on a 1% agarose gel to assess quality and amplification yield. On a good experiment we expect to see a smear, typically between 200 and 1500 bp, on the products from Lamin B1-Dam and Dam alone, and nothing on any of the negative controls. If the negative controls are clear, then the amplified DNA is cleaned up using the Qiagen QIAquick PCR purification kit and the DNA quantified. How much DNA is required for sequencing will depend on the company used to carry it out and the sequencing platform they use. We typically pooled together the products from 4 to 5 PCRs as described above, and purified the DNA together. This way we obtained more than the 2.5 µg that our chosen sequencing facility required. More or fewer PCRs may be required in order to meet the requirements of different sequencing facilities. It is important to keep the number of cycles relatively low in order to avoid excessive bias towards more abundant sequences inherent to PCR. For this reason we do not recommend increasing the number of cycles stated here.2.5We have performed LaminB1-DamID experiments with as few as 20 million 'clean' reads for human or mouse genomes with some degree of success, although we would recommend at least 30–40 million minimum. By clean reads, we mean the number of sequences obtained per sample after filtering contaminants and sequences that map to multiple regions in the genome. Noisy data due to suboptimal sequence coverage makes analysis more complicated and reduces reliability, which makes the use of multiple replicate experiments more important. One way to deal with noise is to apply local smoothing, but that reduces the spatial resolution. There is no substitute for a healthy number of good quality sequence reads to obtain good quality results.2.5.1http://www.bioinformatics.babraham.ac.uk/projects/fastqc). Sequences from each sample were then mapped to the mouse genome mm9 NCBI build 37 using the Burrows-Wheeler Aligner (BWA), and sequences mapping to mitochondria or to ambiguous locations were eliminated The DNA obtained at step 2.4.3 was sent for sequencing offsite. We have generally used Beijing Genomics (BGI), and prepared the DNA according to their specifications. For example, using the Illumina HiSeq 2000 platform in conjunction with 90 bp read lengths, we submitted 2–2.5 µg of DNA per sample. Sequencing libraries were prepared by BGI by randomly fragmenting the DamID DNA and ligating sequencing adaptors to the ends. We have been able to obtain up to 45 million reads per sample, running four samples multiplexed per well. These details will depend on the sequencing platform and facility used and so it is important to calculate expected read depth before engaging sequencing. Some facilities will provide sequence data that have already been filtered to remove low quality reads in addition to the raw data, and some will just provide the raw sequences. Raw data can be easily custom-filtered using the Java tool FastQC . The resulting files can be imported into the Integrated Genome Viewer  for visualization, where LADs appear as regions with positive signal, separated by interLADs. The data can appear ‘noisy’ both when sequence coverage is suboptimal and due to aspects of the biology of the experiment. The 'noise' is obvious when zooming into interLADs which then may appear to have frequent spurious positive signal fragments. The reason the noise is not apparent when zooming out is that DamID profiles appeared smoothed on the screen. The best way to deal with noisy data is to produce better data, but that is not always an option for financial or technical reasons. In that case, it is possible to apply gentle smoothing at the cost of losing some spatial resolution and dynamic range. Considering that the median DpnI fragment size in mouse is 260 bp and that 95% are under 1238 bp, a smoothing factor of 20 would be reasonable, where the value assigned to each DpnI fragment is substituted by the average of the 20 fragments surrounding it. This would lose only a few Kb of resolution but the cleaner profiles make LAD detection easier. It is important to consider that in smoothing the data as indicated, DpnI fragments with high read counts are treated just the same as DpnI fragments with low read counts, whose quantitation is less reliable. While this can work well enough, as illustrated here, there are other alternatives that one could consider. For example, smoothing before normalization or quantifying the reads using bins of a constant size rather than DpnI fragments, can produce good results too and may be less influenced by the mix of high and low read count fragments. Experimenting with different sized bins is recommended, but we find that 2–5 Kb bins is usually a good starting point.For analysis, we quantified 'normalized counts' per genomic 2.5.2LADs can be visually observed using a browser like IGV. LADs are thought to typically range from a few tens of kilobases to several megabases long, and cover up to 50% of the genome. This does not necessarily mean that half of the genome is located at the nuclear periphery of the nucleus at any one time, but it is probably a reflection of the variability in LADs between individual cells in the population 3in vitro muscle differentiation system due to high backgrounds of methylated DNA from mitochondria that accumulate within invaginations of muscle nuclei as well as several other factors The original DamID procedure was developed for use with tissue culture cancer cell lines 3.14/cm2 allowed to reach the confluence and to form a monolayer after 24 h of culture, which is crucial to maintain hepatocyte phenotype and metabolic activity. The original DamID procedure requires maintenance of the cell cultures typically for three days after transduction for sufficient Dam-fusion protein expression and for achieving optimal Dam activity and labeling of specific DNA loci. Hepatocytes isolated for in vitro studies are prone to losing their defined differentiation state progressively with the time of culture, as can be determined by their obtaining a more fibroblast-like aspect by 72 h. To prevent loss of differentiation, at least for the duration of DamID experiments, the culture medium used was optimized for maintaining liver-specific functions of hepatocytes by removing serum and supplementing with insulin, dexamethasone, and EGF, which helped in slowing the dedifferentiation process and in increasing culture longevity in vitro) permitted the correct maintenance/reassembly of chromatin architecture and localization of expressed Dam-tagged protein of interest. The absence of mycoplasma infection was detected by performing a specific PCR amplification protocol on isolated genomic DNA, which ensures enough sensitivity even for low infection levels In order to successfully apply the DamID protocol in primary mouse hepatocytes, a proper isolation technique should be mastered to produce viable cultures. A detailed methodology and equipment description have been published 3.2in vitro, which precludes foreign DNA to access the nuclear transcriptional machinery that for most methods requires nuclear envelope breakdown in cell division. Electroporation-based methods, such as nucleofection, could be used as an alternative for hard-to-transfect cells, although with low efficiencies (<5%) and high toxicity in primary hepatocytes . For this reason, we optimized a lentivirus-based transgene delivery for our DamID purpose, taking advantage of the charge negating agent polybrene and the high transduction efficiency, even in non-dividing cells, of VSV-G pseudotyped lentiviral particles encoding for native Dam or Dam-Lamin B1 fused proteins. Lentiviral transduction was also chosen over transfection because plasmid DNA generated in most routinely used bacterial strains will also harbor the m6A methylation so that its amplification would be likely to outcompete the specifically DamID methyl-tagged sequences from the genomic DNA during the DamID processing of samples. In fact, even Dam plasmids isolated from Dam-negative strains usually harbors detectable amounts of m6A due to leaky expression of the Dam construct. Transduction of both human and murine primary hepatocyte cultures has been already established for basic and clinical studies by using lentiviruses at high titer The DamID technique has the potential to be adapted to any cell line or animal into which the Dam-expressing constructs can be efficiently delivered. The differentiation status of a particular cell type strongly influences the ability of the cell to uptake exogenous DNA. In general, fully differentiated cell types are much more difficult to be genetically engineered than proliferating and undifferentiated cell types. Protocols for applying the DamID method in a variety of immortalized cell lines or primary cells have already been described, but none for primary mouse hepatocytes yet We investigated the effect of the transduction enhancer polybrene in primary hepatocytes by the observation of changes in cellular morphology and metabolic activity. Polybrene has been shown to markedly increase transduction efficiency but it can also negatively affect cell viability at high concentration To evaluate gene transduction efficiency in primary mouse hepatocytes, we obtained a lentiviral vector expressing the fluorescent protein EGFP, under the control of the human phosphoglycerate kinase (hPGK) promoter. Isolated cells were tested for transgene expression after 72 h of transduction with different amounts of highly concentrated lentiviral preparations by Western blot and confocal fluorescence microscopy analyses. Following transduction, hepatocytes were viable and showed that the expression levels of the EGFP reporter protein correlated with the increase of the volume of lentiviral particles added to the culture medium A. MoreovDrosophila minimal heat shock protein promoter available from the van Steensel lab Another important aspect is that expression levels of the Dam fusion proteins should be very low, nearly undetectable by Western Blot or fluorescence microscopy, to avoid the production of a noisy background signal or cellular toxicity as a result of the Dam methylase activity. For this reason, weak promoters for Dam-encoding vectors should be considered like the 3.3Sequencing depth is probably the single major determinant of the quality of the data. The quality and amount of DNA submitted for sequencing plays an important role in ensuring the quality of the sequences obtained. When DNA amounts are suboptimal this can often result in a large number of redundant sequences produced from a single amplicon PCR amplification. This can sometimes be observed as prominent discrete bands within the normal smear, although not always. A normal pattern of amplification is shown in DpnI fragment by the average of n fragments surrounding it. This cleans up the profiles, but it also reduces dynamic range and spatial resolution. The median DpnI fragment size is 260 bp, with the 95th percentile at just over 1.2 Kb, meaning that even smoothing by 20 fragments, we should only be a few Kb off when calculating boundaries, which will typically make little differences as LADs tend to be tens to hundreds of Kb long. Mitochondrial DNA is also sometimes recovered together with genomic DNA. In our hands, mitochondrial sequences can comprise between 7 and 26% of the total number of sequences recovered from primary mouse hepatocytes. The reasons for this variability are not clear to us, but the amount of mitochondrial DNA co-purified with genomic DNA seems to be reproducibly higher in primary cells than in tissue culture cancer cell lines. For example, in LaminB1-DamID experiments performed with human fibrosarcomaHT1080 cells 3.4LAD coordinates are generally determined from DamID profiles  using a peak finding algorithm. There are many suitable for the task, each with their own pros and cons. For example, Spatial Clustering for Identification of ChIP-Enriched Regions (SICER) was created mostly to identify histone modification marks in chromatin, which are generally regions much smaller than LADs and with sharper boundaries in vitro differentiation system that DamID signals exhibit a reasonably strong inverse correlation with their radial distance to the nuclear envelope In order to compare LADs between two or more conditions, the accepted norm has been to use a binary comparison: absence/presence of LAD, to obtain all differential regions. The success of this approach is highly dependent on an accurate determination of all LADs. An alternative approach would be to compare signal intensity differences between conditions within a window of a given length running along each chromosome. The biological meaning of the signal intensity became clearer when the DamID method was adapted to work in single cells 4in vivo spatial nuclear chromatin architecture in mouse liver cells. This methodology could be useful in order to recognize key regulators of hepatic metabolic functions and could be directly applied to assess global changes in chromatin organization and gene positioning in primary hepatocyte cultures derived from liver-specific NETs knockout mice.In this work, we describe a DamID-Seq procedure optimized for LADs identification in primary mouse hepatocytes. This approach has enabled us to profile 51)The presence in the culture medium of supplements like insulin and dexamethasone during the washing steps and culture maintenance helps in slowing the dedifferentiation process typically displayed by isolated hepatocytes.2)The addition of EGF to hepatocyte culture medium induces the cells to undergo at least one cycle of replication to allow monolayer formation and to maintain liver-specifc organization of chromatin architecture. Furthermore, EGF enhances the transduction efficiency and, accordingly, the expression of Dam-Lamin B1 fusion protein to the nuclear envelope, which results in specific DNA labeling by Dam on target DNA sequences.3)Lentiviral preparations could be used at higher titer since they showed low cellular cytotoxicity in primary hepatocytes.4)Due to the high autofluorescence of hepatocytes we suggest to quench PFA fixed samples as described in materials and methods section and more importantly we encourage the indispensable use of a confocal microscope to acquire fluorescence images by finely selecting excitation and emission wavelenghts for EGFP channel.5)It is highly recommended to use up to four wells for each condition when performing genomic DNA extraction for DamID experiments, to ensure the collection of enough material for downstream processing.6)It is important to use the DNeasy blood and tissue kit. In our experience the DamID results are extremely variable when using phenol–chloroform extractions to isolate DNA.


Compared to conventional Ag nanoparticle films, dendritic Ag nanostructures exhibited larger SERS enhancement ascribed to the numerous hot spots exist in the nanogaps of parallel and vertically stacked multilayer Ag dendrites. In addition, the prepared dendritic Ag nanostructures show 3.2-fold higher catalytic activity towards the reduction of 4-nitrophenol (4-NP) by NaBH4 than the Ag nanoparticle films. The results indicate that the dendritic Ag nanostructures represent a unique bifunctional nanostructure that serves as both efficient catalysts and excellent SERS substrates, which may be further employed as a nanoreactor for in situ investigation and real-time monitoring of catalytic reactions by SERS technique.Highly branched metallic nanostructures, which possess a large amount of catalyst active sites and surface-enhanced Raman scattering (SERS) hot spots owing to their large surface areas, multi-level branches, corners, and edges, have shown potential in various applications including catalysis and SERS. In this study, well-defined dendritic silver (Ag) nanostructures were prepared by a facile and controllable electrochemical deposition strategy. The morphology of Ag nanostructures is controlled by regulating electrodeposition time and concentration of AgNO Noble metal micro/nanostructures have attracted great attention due to their potential applications in optics , catalysThe applications based on the substrates with plasmonic nanostructures have been extensively explored , 7. Most3 concentration, deposition time, deposition current density, and citric acid concentration were systematically investigated to reveal the influences of AgNO3 concentration and deposition time on final morphologies. The prepared dendritic Ag nanostructures exhibited larger SERS enhancement and catalytic activity compared to the Ag nanoparticle films prepared by magnetron sputtering method.In this work, dendritic fractal nanostructures on indium tin oxide (ITO) glass substrates were fabricated by a facile and controllable electrochemical deposition strategy. The shape evolution induced by the AgNO2, 17 Ω/square) cathode and a platinum (Pt) plate anode. ITO glasses were cleaned by ultrasonication in acetone, distilled water, and ethanol for 15 min, respectively. The distance between the two electrodes was set to be 3 cm. The electrolyte solution contained AgNO3 (2 g/L) aqueous solution and citric acid (40 g/L). In the electrochemical deposition process, a constant current density of 1 mA cm−2 was applied. After the electrodeposition process was completed, the samples were rinsed with ultrapure water for several times and then dried with high-purity flowing nitrogen. The as-electrodeposited dendritic Ag fractal nanostructure samples were then submerged into 10−5 M 3,3′-diethylthiatricarbocyanine iodide (DTTCI) ethanol solution for 4 h to adsorb a self-assembled monolayer of molecules. The SERS samples were carefully rinsed with ethanol to remove the weakly bound molecules and then dried under N2 before analysis.Dendritic Ag fractal nanostructures were prepared by an electrochemical deposition process, which is described in our previous work , 25. The−5 M) aqueous solution was mixed with 1 mL of ice-cold NaBH4 (6 × 10−2 M) aqueous solution under magnetic stirring conditions. A piece of catalyst (the obtained dendritic Ag nanostructure sample and Ag nanoparticle films) with the size of 5 × 10 mm2 was added into the reaction mixture. The reducing process of 4-NP was monitored by measuring the absorption spectra of the reaction solution at regular intervals.In a typical 4-nitrophenol (4-NP) reduction reaction, 1 mL of 4-NP (2 × 10λ = 1.5406 Å). The time-dependent absorption spectra of the reaction solution were measured using an UV-Vis spectrophotometer (TU-1810). SERS spectra were measured by using a micro-Raman spectrometer (HORIBA Jobin Yvon LabRAM HR800). The SERS samples were excited by focusing a 488-nm laser beam onto the sample through a × 50 objective.The structure of the electrodeposited Ag products was characterized by using transmission electron microscope  and scanning electron microscope  equipped with an energy-dispersive X-ray spectrometer (EDX). X-ray diffraction (XRD) measurements were performed on a Bruker D8-advance X-ray diffractometer with Cu Kα1 irradiation , four morphologies such as meatball-like nanoparticles , only some flower-like nanoplates were formed and Ag dendrites were seldom observed , the dendrites greatly extended at the lateral and vertical orientations to form a large-sized “fern-leaf” spread on the ITO glass surface . Under a low deposition current density (0.5 mA cm−2), only some micro-hemispheres grew on the ITO glass surface , the coexistence of Ag dendrites and nanoparticles was found on the ITO glass surface  and current density (1 mA cm−2), without citric acid in the electrolyte, only irregular micro-particles (without any dendrites) were obtained on the ITO glass surface (Fig. The effects of citric acid concentration on electrodeposited products were also explored. Under the fixed AgNOace Fig. a, indicaace Fig. c. When tace Fig. b, d.Fig.3 concentration, deposition time, deposition current density, and citric acid concentration. Obviously, the whole growth process is a non-equilibrium state as the fast nucleation and growth contribute to the formation of more complicated structures  versus time for the reduction of 4-NP catalyzed by dendritic Ag nanostructures and Ag nanoparticle film are shown in Fig. k of the reaction catalyzed by dendritic Ag nanostructures, and Ag nanoparticle film were calculated to be 2.88 × 10−2 and 0.91 × 10−2 min−1, respectively. The reaction rate of the dendritic Ag nanostructures was about 3.2 times higher than that of the Ag nanoparticle film. The large surface area and more active sites are two rules in the design of catalysts. The dendritic Ag nanostructures exhibited the higher catalytic performance because the dendritic Ag nanostructures had a hierarchical fractal structure with large surface areas and many multi-level branches, corners, and edges, which provide a large amount of “catalyst active sites.” We thus believe that the dendritic Ag nanostructures have potential applications in catalytic reactions.We used the reduction reaction of 4-NP by NaBH−5 M ethanol solution of DTTCI on the dendritic Ag nanostructures and the Ag nanoparticle film at 488 nm laser excitation. When DTTCI is adsorbed on the dendritic Ag nanostructures, a large Raman signal is obtained, which is attributed to the DTTCI molecules [−1 is utilized to compare the SERS intensity. The SERS signal of DTTCI molecules on the dendritic Ag nanostructures sample is ~ 30 times stronger than those on the Ag nanoparticle film. Ten randomly chosen spots on dendritic Ag nanostructure sample and Ag nanoparticle film were used to calculate the enhancement factor by counting the intensity ratio of SERS signal. Such large enhancement could be attributed to the fact that more hot spots with largely enhanced localized field were formed at the nanogaps of parallel and vertically stacked multilayer Ag dendrite “film.”Furthermore, DTTCI was chosen as the analyte molecule to investigate the SERS performance of the dendritic Ag nanostructures. Figure olecules . The str3 concentration and electrodeposition time were the key parameters of the formation of well-defined dendritic Ag nanostructures. Dendritic Ag nanostructures exhibited larger SERS enhancement and higher catalytic activity than Ag nanoparticle films. The excellent SERS performance and high catalytic activity should be ascribed to the high-density SERS hot spots and catalyst active sites provided by the large surface area, numerous branches, tips, edges, and gaps of dendritic Ag nanostructures. This work provides a simple route for large-area and shape-controlled synthesis of dendritic Ag nanostructures as an effective catalyst and excellent SERS substrate, which may have great potential in in situ SERS investigation and monitoring of catalytic reactions.In conclusion, we have prepared the dendritic Ag nanostructures by a facile and controllable electrochemical deposition method. AgNO


Moreover, by integrating PrimerROC with our PrimerDimer prediction software we can determine a ΔG-based dimer-free threshold above which dimer formation is predicted unlikely to occur. Notably, PrimerROC determines this cut-off without any additional information such as salt concentration or annealing temperature, meaning that our PrimerROC method is an assay and condition independent prediction tool. To demonstrate the broad utility of PrimerROC we assessed the performance of seven publically available primer design and dimer analysis tools using a dataset of over 300 primer pairs. We found that our PrimerROC/PrimerDimer software consistently outperforms these other tools and can achieve predictive accuracies greater than 92%. To illustrate its predictive power this method was used in multiplex PCR design to successfully generate four resequencing assays containing up to 126 primers with no observable primer-primer amplification artefacts.To-date systematic testing and comparison of the accuracy of available primer-dimer prediction software has never been conducted, due in part to a lack of tools able to measure the efficacy of Gibbs free energy (ΔG) calculations at predicting dimer formation in PCR. To address this we have developed a novel online tool called PrimerROC ( Thus, accurate dimer prediction algorithms are of great value when designing primers for polymerase-based applications, such as DNA sequencing, polymerase chain reaction (PCR), and a variety of isothermal amplification methods. Accurate prediction is especially important in applications such as real-time PCR, where poor amplification efficiency and off-target artefacts interfere with accurate quantification; as well as multiplexing, where the potential for dimer formation increases polynomially, according to the function (n2 + n)/2, with each primer (n) added to the multiplex reaction2.Primer design software seeks to maximize product yield and minimize off-target amplification, and a key component of this is the prevention of the primer-primer interaction artefacts known as primer-dimers. Primer-dimers are off-target amplification artefacts formed by primer-primer binding and subsequent elongation. These primer-primer interactions can competitively inhibit binding to target DNA, remove primers from the reaction pool, and exhaust deoxynucleotides—all of which result in reduced amplification efficiency and suboptimal product yields7. However, in our assessment of publically available tools, each program calculates ΔG differently, and to-date no studies have empirically demonstrated the efficacy of ΔG in general—or specific algorithms in particular—at predicting primer-dimer formation. While some of these programs offer the ability to alter temperature and PCR component concentrations for ΔG calculations , it is unclear how this aids users in determining the likelihood that a particular ΔG value will result in the formation of dimer artefacts. As such, a method for accurate dimer prediction which implicitly normalizes assay-specific prediction values for dimer formation based on a user’s unique conditions would be of significant use to the field. In response to this, we have therefore developed PrimerROC— an application that employs receiver operating characteristic (ROC) curves in a novel way to test predictive power and provide measures of dimer likelihood (sensitivity and specificity) regardless of the unique PCR conditions used.Numerous primer design programs use the change in Gibbs free energy (ΔG) resulting from primer hybridization as an absolute indicator of dimer formation8. Here we use PrimerROC analyses to evaluate and iteratively improve the performance of our ΔG-based dimer-prediction algorithm, PrimerDimer. We then integrate PrimerDimer into PrimerROC to produce an online tool that allows users to distinguish between dimer-forming and dimer-free primer pairs to a high degree of accuracy (>92%). Performance is then compared to seven other commonly used primer design/analysis tools and PrimerROC consistently showed superior predictive power and greater ability to produce dimer-free discrimination thresholds across a variety of primer lengths. Finally, we demonstrate that the high accuracy of PrimerDimer and the dimer-free threshold derived from PrimerROC analysis can be used to prevent dimer formation in highly multiplexed PCR/resequencing reactions.ROC curves plot the performance of a diagnostic marker as a binary classifier. Each marker value is set as a discrimination threshold, the cut-off below which values are classified as condition positive and above which values are classified as condition negative. The true positive rate (sensitivity) and false positive rate (1-specificity) at each threshold value are then plotted against one another to create the ROC curve. The area under the curve (AUC) provides a measure of overall predictive accuracy (1 being perfect and 0.5 is no better than chance), and ROC curves are regularly used in clinical epidemiology to quantify the accuracy of medical diagnostic tests10. Surprisingly, both 3′ ends do not have to form a continuous stable structure for exponential amplification to occur. Within the sequenced dimers, stable structures at a single 3′ end regularly formed amplification artefacts of high concentrations, although it was also observed that 5′ overhangs on the side of the stable structure were often duplicated in the resulting dimer artefact , after which the most negative ΔG-based value is then returned as the dimer score . The shorter primer slides along the longer primer to form all possible dimer structures with 5′ overhangs. The ΔG of each possible dimer structure is calculated using nearest-neighbour parameters for duplexesT) values of dimer-free versus non-extensible dimer forming primer pairs  that is independent of primer length and GC content . However, other conditions cannot be adjusted for, such as pH or the addition of PCR enhancers .The PrimerROC method is condition-independent, in that the only input required from the user is their primer pair sequences and dimer-forming status. This allows users to not only determine a dimer-free threshold under varying conditions but also to assess how well the PrimerDimer algorithm performs under the specific conditions used. Dimer prediction algorithms cannot perform equally well under all conditions, as there are conditions that cannot be adjusted for in the calculation of ΔG. The nearest-neighbour thermodynamic parameters used by dimer prediction tools were determined under the specific buffer conditions of 1.0 M NaCl, 10 mM sodium cacodylate, and 0.5 mM NA2) or formamide . The overall number of dimers formed, as well as which specific primer pairs formed dimers, varied under each condition . We used an annealing temperature of 57 °C in the shorter 2 and 3 base pair fusion sets, and 60 °C annealing/extension for the first 15 cycles of the 14 and 20 base pair fusion sets. The remaining 20 cycles were run at 65 °C, as fusion sequences do not bind to genomic/bisulfite DNA at the primer’s target site but do bind to subsequent amplicons after the initial PCR cycle. To test the ability of PrimerROC to accurately predict dimers under varying PCR conditions we ran the first 48 primer pairs of the 14 base pair fusion set with a lower 57 °C annealing temperature and added the PCR enhancers tetramethylammonium chloride . Notably, changing the temperature and salt conditions when calculating ΔG to better reflect those of the PCR conditions used had a substantially negative impact on predictive performance compared to the standard ΔG°ium Fig. .Figure 9Most of the tools tested use the standard 37 °C in 1 M sodium to calculate ΔG. However, NetPrimer and PerlPrimer allow users to input the temperature at which ΔG is calculated. We, therefore, tested these tools at their default 25 °C, the standard 37 °C, and a high 57 °C when determining the ΔG for the longer 14 and 20 base pair fusion sets to see how this would impact their predictive performance. Overall, the effects of temperature were minor in both tools, although PerlPrimer was better able to produce a dimer-free threshold at the lower 25 °C. Additionally, the input of cation concentrations also influence the calculation of ΔG in PerlPrimer. We, therefore, tested the standard 1 M sodium as well as the sodium and magnesium concentrations used in our PCR runs  to see how cation concentration altered predictive performance. Performance peaked at 25 °C at 1 M for the 14 base pair set and 25 °C at 320 M for the 20 base pair set. The combined effects of temperature and cation concentration were minor on AUC, but did influence the true negative rate and whether or not a dimer-free threshold was established Fig. .Figure 119, and combined this with a modified version of our multiplexing tool PrimerPlex, to produce a multiplex design procedure that minimizes the likelihood of primer-primer elongation for each new region added to the growing multiplex pool. Using this procedure we produced four PCR/resequencing multiplex reactions each with between 43–63 primer pairs with no visible dimer products  for the most stable structure of each primer pair was entered into a spreadsheet along with the forward and reverse primer sequences and whether dimer artefacts were observed in gel electrophoresis of both template and template-free PCR runs. Bands where it was unclear whether they were the result of extensible or non-extensible dimers, including artefact bands that were extremely faint in both template and template-free lanes, appeared in only the template-free but not the template lane, or were very small but dark when compared to amplicon bands, were excluded from analysis. Due to the high number of ambiguous bands that appeared on the first run of the 20 base pair fusion primer set, PCR and gel electrophoresis was performed an additional two times in order to resolve these ambiguities. Primer tables were then loaded into an in-house ROC generating python script to create ROC curves reporting the AUC and dimer-free discrimination threshold. Where programs reported both most stable overall and 3′-dimer structures, we assessed the ability of both these scores to predict dimer formation and reported results of the method with greater performance.Used PerlPrimer version 1.1.21 to assess dimer prediction. The PCR component concentrations under the General tab were set to the conditions used in the Bisulfite PCR Conditions section below . Forward and reverse primer sequences were input under the Primers tab to calculate the ΔGs of the most stable homodimer and heterodimer structures. The temperature for ΔG calculation was set to 37 °C under Preferences > Dimers to standardize with other programs that offer a static 37 °C without the option to alter the temperature parameter. In addition, ΔGs were also calculated at PerlPrimer’s default 25 °C, as well as 57 °C, for the 14 and 20 base pair fusion sets to see if changing the temperature parameter to calculate ΔG increased predictive power. Each temperature was also paired with the cation conditions listed above , as well as 1000 mM monovalent cations (0 mM Mg++) ROC curves were generated for both the most stable non-extensible primer-dimers and most stable 3′ extensible primer-dimers values using the most negative ΔG for each primer pair.Used Oligo 7 version 7.60 (DEMO) to assess dimer prediction. Forward Primer and Reverse Primer sequences were input under the Edit menu and the ΔGs of the most stable overall and 3′-dimer of the Forward Primer, Reverse Primer, and Mixed Oligos were recorded from Analyze > Duplex Formation. ROC curves were generated for both the overall and 3′-dimer values using the most negative ΔG for each primer pair. Oligo 7 does not offer the options to alter temperature or cation concentration in the calculation of ΔG.Used OligoAnalyzer version 3.1 to assess dimer prediction. The PCR sets were set to the conditions used in the Bisulfite PCR Conditions section below . However, these parameters do not appear to influence the resulting ΔG values and are only used in the calculation of melting temperature. Forward and reverse primer sequences were input separately to determine the ΔGs of the most stable homodimers using the Self-Dimer function. The most stable heterodimer structure was then determined using the Hetero-Dimer function. OligoAnalyzer does not offer the option to alter temperature.Used MFEprimer version 2.0 to assess dimer prediction. The Experimental settings were set to the conditions used in the Bisulfite PCR Conditions section below . Forward and reverse primer sequences were input to determine the ΔGs of the most stable homodimer and heterodimer structures. MFEprimer 2.0 does not offer the option to alter temperature.Used NetPrimer to assess dimer prediction between 24 March 2016 and 22 April 2016. The Reaction Conditions were set to the conditions used in the Bisulfite PCR Conditions section below . However, these parameters do not appear to influence the resulting ΔG values and are only used in the calculation of melting temperature. Forward and reverse primer sequences were input to determine the ΔGs of the most stable Self Dimer (homodimer) and Cross Dimer (heterodimer) structures. The Temperature for Free Energy Calculation was set to 37 °C to standardize with other programs that offer a static 37 °C without the option to alter the temperature parameter. In addition, ΔGs were also calculated at NetPrimer’s default 25 °C, as well as 57 °C, for the 14 and 20 base pair fusion sets to see if this increased predictive power. ROC curves were generated using both the minimum (most negative) ΔG and minimum 3′ Dimer ΔG for each primer pair.Used Multiple Primer Analyzer to assess dimer prediction between 24 March 2016 and 30 May 2016. The Parameters for calculation of primer Tm were set to the conditions used in the Bisulfite PCR Conditions section below . However, these parameters do not influence the presence of dimers at different sensitivity settings and are only used in the calculation of melting temperature. Forward and reverse primer sequences were input in FASTA format and the Value of the sensitivity for dimmer detection was sequentially raised from the maximum sensitivity (1) to the minimum sensitivity (10). The sensitivity before which dimer structures were no longer present in the ‘Results for primer-dimer detection’ output box was then recorded for each primer pair. Less stable structures require more sensitivity to detect, meaning the most stable structures remain the longest as the sensitivity is lowered. If no dimers were present at the maximum sensitivity of 1 the value recorded was 0. Multiple Primer Analyzer does not offer the option to alter temperature.Used Primer3 version 2.3.7 to assess dimer prediction. Primer3 was installed on our Linux server via the instructions presented in the Primer3 Release 2.3.7 Manual. Forward and reverse sequences were pasted into the inputfile.txt and primer3_core was then run on this file. The most negative overall ΔGs for homodimers and heterodimers, as well as the most negative 3′ ΔG for homodimers and heterodimers, were then retrieved from the stdout. Primer3 does not offer the options to alter temperature or cation concentration in the calculation of ΔG.https://www.python.org), and later adapted into a web application using the Django framework (version 1.8+) (https://www.djangoproject.com), and hosted via Apache2 HTTP server (https://httpd.apache.org) on the nectar cloud (https://nectar.org.au).The PrimerROC/PrimerDimer software was originally written in the Python language (version 3.4+)  to create all possible dimer structures with 5′ overhangs, with each end treated independently.11 and single mismatches16, or assigns a penalty for double mismatches. The NN ΔGs of subsequent nucleotide pairs are then progressively added to determine the dimer structure’s ΔG. The most negative ΔG derived from a single NN iteration of this process is taken as the dimer score. Next, penalties are added for single base mismatches in the first few bases from the 3′ end of the dimer structure, and a further penalty is added if either the first or second base is a mismatch in addition to at least one other mismatch in the first 5 bases. The ΔGs of 5′ overhangs17 are then added and finally, a bonus is added if the first base of the dimer structure is a C or G and the structure spans both 3′ ends with 100% of base pairs matched. All bonuses and penalties were derived by iterating through a series of values for each parameter on the 3 and 20 base pair training sets. Those values that gave the maximum dimer-free true negative rate  were used in the final algorithm.PrimerDimer starts from the first two bases at the 3′ end and determines their ΔG  using nearest-neighbour (NN) parameters for duplexesThe dimer score (dS) length/GC adjustment was also derived from this method. The pseudocode for the calculation of the length/GC adjusted dS is detailed below:This procedure iterates through a list of input DNA regions, producing all viable forward and reverse primer pairs. Regions are then sorted from those with the least to those with the most primer pairs. Prospective primers for each region are paired with all other primers within the existing pool and the most negative ΔG-based dimer score of all these interactions is recorded. If the score is within the dimer-free range, the primer pair is stored as a candidate for its particular region. The primer pair with the highest (least negative) dimer score for a region is then added to the growing multiplex pool.20. For each conversion, DNA was first quantified with the Qubit dsDNA BR Assay Kit and, based on the available sample material, between 100 ng – 1 µg of material was bisulfite converted at a time. Conversion took place at 80 °C for 45 minutes, followed by resuspension in low TE .Bisulfite conversion of template DNA was conducted using manual protocols reported previouslyet al.21 for CES recipe); MgCl2 ; dNTP’s ; primers ; Taq ; DNA . Amplification took place on either an Eppendorf ProS 96 well, or Eppendorf Pro 384 well thermocycler. Cycling conditions were as following: Primers with 2 and 3 base pair fusion sequences: 35 cycles ; Primers with 14 and 20 base pair fusion sequences: 15 cycles ; and 20 cycles . All reactions started with a 10 min 95 °C step for activation of the Hot-Start GoTaq® DNA Polymerase and finished with a 10 °C hold. PCR products were evaluated using standard 2% agarose gel electrophoresis techniques with SB buffer and ethidium bromide. Each primer pair was screened against two samples: a bisulfite–converted DNA template and no template control. The three altered reagent and cycling conditions for the 14 base pair fusion set were the same as those of the 2 and 3 base pairs set conditions detailed above with the following differences: (1) 1.5 mM TMAC, 4 mM MgCl2; (2) 1.5% formamide, 6 mM MgCl2, no CES; (3) 4% formamide, 6 mM MgCl2, no CES.A PCR mastermix recipe for amplification of bisulfite-converted DNA was made and the final PCR reaction had the following components at the indicated concentrations: 5X Promega GoTaq; 5X Green Flexi Buffer ; CES 5 ×  and ROX reference dye for real-time PCR , and replacement of 5X Green Flexi Buffer with 5X Colorless Flexi Buffer. Non-extensible dimers were determined by their appearance as bands in three separate samples on ethidium bromide stained gel electrophoresis. These samples included: a bisulfite–converted DNA template, no template control, and Taq-free control.2; 200 µM dNTPs; 0.05 U/µL HotStart Taq. DNA from each reaction was then purified using Agencourt XP bead cleanup and individually amplified with Ion Xpress™ Barcode Adapters. Cycling conditions were: 95 °C, 10 mins; 15 cycles ; 68 °C, 2 mins; 4 °C, 5 mins. Barcoded PCR products were then pooled and excess primers were extracted using gel purification (Promega gel cleanup kit). Amplification took place on either an Eppendorf ProS 96 well, or Eppendorf Pro 384 well thermocycler.Initial amplification cycling conditions were: 95 °C, 10 mins; 15 cycles of ; 72 °C, 2 mins; 4 °C, 5 mins. The final concentrations for each reaction were: 1x Promega GoTaq Green Flexi buffer; 4.5 mM MgClSequencing was performed on the Ion Proton™ System. Template preparation was performed with the Ion OneTouch™ 2 System (OT2) using the Ion PI™ Hi-Q OT2 200 Kit. Proton runs used the Ion PI™ Chip Kit v2 and Ion PI™ Hi-Q Sequencing 200 Kit.Dimer artefacts were derived from no-template controls and barcoding was performed using commercially purchased oligonucleotides with MiSeq sequencing adaptors and barcodes (Fluidigm PN FLD-100-3771). Sequencing was performed on the Illumina Miseq. MiSeq runs used the MiSeq Reagent Kit v2, 300 cycle; PN MS-102-2002.22 version 2.3.0-legacy for genomic and Bismark23 running Bowtie 2  for bisulfite converted DNA. Graphing and statistical analysis employed IBM SPSS Statistics 24.Adaptor trimming used Trim galore . Mapping of sequenced data used Bowtie 2Supplementary Info


RBMY1 genes are located in four variable-sized clusters on the Y chromosome, expressed in male germ cells and possibly associated with sperm motility. We have re-investigated the mutational background and evolutionary history of the RBMY1 copy number distribution in worldwide samples and its relevance to sperm parameters in an Estonian cohort of idiopathic male factor infertility subjects. We estimated approximate RBMY1 copy numbers in 1218 1000 Genomes Project phase 3 males from sequencing read-depth, then chose 14 for valid ation by multicolour fibre-FISH. These fibre-FISH samples provided accurate calibration standards for the entire panel and led to detailed insights into population variation and mutational mechanisms. RBMY1 copy number worldwide ranged from 3 to 13 with a mode of 8. The two larger proximal clusters were the most variable, and additional duplications, deletions and inversions were detected. Placing the copy number estimates onto the published Y-SNP-based phylogeny of the same samples suggested a minimum of 562 mutational changes, translating to a mutation rate of 2.20 × 10−3 (95% CI 1.94 × 10−3 to 2.48 × 10−3) per father-to-son Y-transmission, higher than many short tandem repeat (Y-STRs), and showed no evidence for selection for increased or decreased copy number, but possible copy number stabilizing selection. An analysis of RBMY1 copy numbers among 376 infertility subjects failed to replicate a previously reported association with sperm motility and showed no significant effect on sperm count and concentration, serum follicle stimulating hormone (FSH), luteinizing hormone (LH) and testosterone levels or testicular and semen volume. These results provide the first in-depth insights into the structural rearrangements underlying RBMY1 copy number variation across diverse human lineages.Human  RBMY gene family was first reported in 1993 under the name Y chromosome RNA Recognition Motif as a group of three or more genes located on the long arm of the human Y chromosome, expressed specifically in testis and deleted in two oligospermic individuals  led to mice with 46% abnormal sperm that were nevertheless fertile  values of 9, 10.7 and 8.7, respectively. This natural variation in copy number allowed the relationship between RBMY1 copy number, RBMY protein level and sperm phenotypes to be investigated, leading to a report of positive correlations between copy number, protein level and sperm motility , ‘2’ , ‘3’  and ‘4’ , from proximal to distal  in region ‘4’. No variation was seen in region ‘3’. The pattern of variation was confirmed by manual review of the log2 ratio of array CGH intensity plots . These initial ratios ranged from 0.62 to 1.89 suggesting that the copy number ranged between 3 and 12, with a broad distribution across the samples. These findings confirm previous work  in the RBMY1 regions of the eight samples as well as the different copy number between three sister pairs of samples in the 1000 Genomes Project phylogenetic tree  where we identified a total of five gene clusters, resulting from a partial duplication of region ‘1’ identified using probe P4   have only a single copy of the gene  . This mithe gene  and 4A.RBMY1 numbers for four sets of different samples; for example, both HG01699 and HG02224 have a total of 10 gene copies, but the underlying structure of the gene regions is different , suggesting that the copy number of RBMY1 genes can be accurately inferred from this correlation. When the copy numbers of the remaining 6 of the 14 fibre-FISH samples were estimated using this correlation, and compared with the measured fibre-FISH number, 5 of the 6 corresponded precisely, and 1 sample differed by one copy (10 versus 11 copies). Although the number of samples tested is low, these results suggest that copy number estimates are generally reliable but can be slightly less accurate at higher copy numbers.The relative total read depths of the four regions combined (compared with an adjacent unique region) were highly correlated with the total number of RBMY1 gene copy numbers were then estimated from the read depth for 1218 1000 Genomes Project male samples using the calibration described above. The estimated RBMY1 copy numbers ranged from 3 to a total of 13 copies , with 8 copies being the most common, found in 430 (35.3%) samples and 7 the second most common, found in 237 (19.5%) ( (19.5%) . It is nRBMY1 copy number changes by taking advantage of the published robust single-nucleotide variant (SNV)-based Y phylogeny available for the same individuals (−3 (95% CI: 1.94 × 10−3 to 2.48 × 10−3) mutations per father-to-son Y transmission  mutations per father-to-son Y transmission . Co. CoRBMY1ividuals . We counsmission .Such a high mutation rate of copy number changes is supported by the three pairs of phylogenetically closely related samples  chosen for fibre-FISH that showed differences in estimated copy numbers from read depth . In all −2 per father-to-son transmission (95% CI: 3.3 × 10−4 to 6.8 × 10−2). Given the low number of father–son pairs available and the identification of only one mutation, the CI is very wide but we see the measurement as a validation of our estimate from the tree.Among 60 father–son pairs in the Complete Genomics data also available for the 1000 Genomes Project samples , we founRBMY1 copy number change is evolving under selection, we developed a different approach that took advantage of the robust phylogeny. Events of copy number increase and decrease for which the direction of change could reliably be inferred were counted in the 1000 Genomes Y phylogeny following the concept of maximum parsimony. We reasoned that if either higher or lower copy number has had a selective advantage in recent human evolution over the 190 000 years covered by this phylogeny, then we should see a bias towards survival of mutations that increase or decrease the copy number, respectively. It was previously reported that increased copy number of RMBY1 is associated with increased sperm mobility, so we would expect to see more increase events if there is selection for higher motility. To our surprise, however, we observed significantly more decrease events  (P = 0.07), perhaps due to the low number of total events. We then added increase (P = 0.06), so there is no evidence for selection for either increased or decreased copy number of the gene. However, the variance of the RMBY1 copy number in the 1000 Genomes samples (1.65) lies below the 0.5th percentile of the variance distribution of 1000 neutral simulations (95% CI: 6.46 to 66.52) using the same number of mutation events on the same phylogenetic tree; this, in the simplest interpretation, suggests that very low and very high copy numbers of RMBY1 gene are selected against.It is not possible to apply standard neutrality/selection tests e.g.  for Y-ch= 0.015) . As we a= 0.015) , again mincrease  and decrincrease  events fincrease  and Polaincrease , but theRBMY1 genes and to investigate the structural variability of different RBMY1 gene copies. We chose a T/C paralogous sequence variant that in the human reference sequence distinguishes the four proximal gene copies in regions ‘1’ and ‘2’, called the ‘T-assay’ from the two distal copies in regions ‘3’ and ‘4’, called the ‘C-assay’. The total copy numbers from T- and C-assays are highly correlated with the measurements from the 14 fibre-FISH experiments (‘gold standards’) , and the difference between the two methods was less than 0.8 copies  of samples carrying two copies of RBMY1.We next typed 376 Estonian idiopathic infertility subjects carrying Y lineage N3-M46, one of the most frequent Y lineages in Estonia, present in ~31% of both the general population  and the RBMY1 estimated from the C-assay shared a sub-lineage of N3-M46 (N3a3a-L550), suggesting a single rearrangement event, potentially resulting from a gene duplication. Such an example is seen in HG02224, carrying three copies according to the C-assay and a total of three gene copies in regions ‘3’ and ‘4’ according to fibre-FISH (Five samples in the cohort with three copies of bre-FISH . A sixthRBMY1 copy number (sum of T- and C-assays) among the Estonian subjects ranged from 5 to as high as 16, with a mode of 10 (247 or 65.7% of samples), showing high levels of variation across the haplogroup N3a phylogenetic tree , hormonal levels  and testicular and semen volume  either, although the reduction in sperm production might in principle cause a secondary decrease in sperm motility , equal to 10 (n = 229) and more than 10 (n = 76)  (n = 246) and normal  progressive motility did not differ for their RBMY1 copy number distribution   . Secondlum test) . AzoospeRBMY1 copy number was not associated with any of the tested parameters in the Estonian idiopathic infertility cohort analysed.Taken together, we find that RBMY1 gene family within the general population, how this arises by mutation, how it may be shaped by selection, and, by studying subjects with unexplained spermatogenic failure, how it may influence sperm phenotypes. We now discuss each of these topics.In this study, we have investigated the variation in the structure and copy number of the RBMY1 copy number  to the genes in the other clusters, perhaps as a result of gene conversion from region ‘1’ or ‘2’ or recurrence of the initial duplication event. While a complete understanding of the complexities of this gene family will require future improved de novo assemblies using accurate long reads, we already see from the fibre-FISH results how frequent increase or decrease in copy number within a tandem cluster, moderately frequent inversion events between adjacent pairs of clusters and possibly less frequent transfer of information between more distant clusters can explain the patterns of sequence identity observed in the reference sequence and the variation in the population.The high level of variation in  to TSPY ) has bee to TSPY , Denmark to TSPY , China . Our second approach was to take advantage of the simple inheritance pattern of the Y chromosome and count the copy number changes in the Y phylogenetic tree, leading to a rate of 2.20 × 10−3 (95% CI: 1.94 × 10−3 to 2.48 × 10−3) mutations per father-to-son transmission. This estimate has not been corrected for recurrent mutations that are likely to have occurred on long branches, so is a minimum estimate, but even so reveals a high rate comparable to Y-STRs  that showed duplication of the whole AZF region  chosen for fibre-FISH experiments from the 1000 Genomes Project were purchased from NHGRI Sample Repository for Human Genetic Research at the Coriell Institute for Medical Research.RBMY1 genes (~14 kb in length) were covered by three PCR products . Two other probes were designed to hybridize outside of the RBMY1 genes: probe P4 to around 42 kb downstream of region ‘1’ to distinguish regions ‘1’ and ‘2’ and probe P5 ~27 kb downstream of region ‘3’ to distinguish regions ‘3’ and ‘4’. The BAC clone RP11-95B23 was used to highlight the target region. Briefly, the BAC DNA and the PCR products were amplified and labelled with GenomePlex Whole Genome Amplification Kits  as described in  . The RBMribed in . RP11-95ribed in . The ordRBMY1 regions and (y) the accurate copy numbers of RBMY1 genes from fibre-FISH were calculated in Excel. We used 8 out of 14 samples with accurately measured copy number from the fibre-FISH experiments, including the sample with 2 copies of RBMY1 in region 4, for the initial correlation calculation, and the remaining 6 to test the accuracy of the inferred copy numbers using the correlation formula.The linear correlation between (x) the sum of read depth ratios of the four RBMY1 copy number change events among all 1218 samples analysed was 562, and there were 371 events among the manually curated set of 839 samples.The published detailed phylogenetic tree  was usedet al. 2018  SNV mutations per bp per year was used (−9 × 10.3 × 107)−1. This was converted into generations assuming a 30 year generation time (−3 (95% CI: 1.94 × 10−3 to 2.48 × 10−3) and 1.78 × 10−3 (95% CI: 1.57 × 10−3 to 2.02 × 10−3) mutations per father-to-son Y transmission, respectively. The confidence interval of the SNV mutation rate was used to obtain the confidence interval for the copy number change mutation rate.A similar approach to Teitz al. 2018  was usedible DNA . Removalwas used . A totalion time . Each SNRBMY1 genes of eight as the ancestral state. We then determined the final copy number of RBMY1 genes at each tip of the tree taking into account the changes in the each simulation, and we treated negative copy numbers as zero. Finally, we calculated the variance of the RBMY1 copy numbers from each of the 1000 simulations and compared this with the empirical variance of RBMY1 copy number changes. The simulation was done using a custom R script.First, we identified confident mutation changes from our real data by counting the ones on the phylogeny where the ancestral state was clear (176 events) and then scaled them up to the total number of mutations (562 events). In this way we inferred ~227, 54, 16, 13 and 3 events for 1, 2, 3, 4 and 5 copy number decreases, compared with 185, 42, 19 and 4 for corresponding increases. These changes were then randomly assigned on the branches of the same phylogenetic tree according to the branch length in each of the 1000 simulations . We assumed the most common copy number of https://github.com/Illumina/Polaris) high-coverage whole-genome sequence data were mapped to GRCh38 and processed using the standard WSI pipelines. Initially, samples overlapping with the 1000 Genomes Project dataset were excluded, leaving a total of 189 unique samples. A total of 16 samples overlapping with the 1000 Genomes Project were included as internal controls. Thus, a total of 205 samples were used for downstream analysis. The RBMY1 copy numbers for these samples were estimated as described above for the 1000 Genomes Project data.The SGDP , the HGDhttp://tree.bio.ed.ac.uk/software/figtree/) with midpoint rooting.The genotypes of the 10.3 Mb of chromosome Y sequence previously defined as accessible to short-read sequencing  were joiAZFa, AZFb, AZFc).The study was approved by the Ethics Review Committee on Human Research of the University of Tartu, Estonia  and genotyping at the Wellcome Sanger Institute under WTSI HMDMC 17/105. Informed consent was obtained from all subjects included in the study. The study subjects were collected at The Andrology Centre at Tartu University Hospital during the period from 2003 to 2015. The collection of samples, definition of causal factors for severe male factor infertility and idiopathic infertility, semen analysis and hormone assays have previously been described in detail . BrieflyAZFc partial deletions following a published protocol ; cryptozoospermia, n = 26 (spermatozoa count of <1 million/ejaculate); severe oligozoospermia, n = 107 (spermatozoa count of 1–10 million/ejaculate) and moderate oligozoospermia, n = 216 (spermatozoa count of 10–38 million/ejaculate).In total, 1190 Estonian idiopathic azoo- and oligozoospermic subjects were initially typed for Y-chromosomal protocol . Among togroup N ,40. Due ogroup N  , which in the human reference sequence (GRCh37) is shared by four RBMY1 copies in the proximal regions ‘1’ and ‘2’. The second probe is specific to the C-allele (C-assay) shared by two gene copies in the distal regions ‘3’ and ‘4’ in the reference sequence. To increase the specificity of the allele-specific ddPCR assays, unlabelled oligonucleotides identical to the probes were used in both assays  .PCR primers and probes  for ddPCken from . Two indRBMY1 genes. PCR reactions were prepared according to the manufacturers recommendations, containing the final concentrations of 1X ddPCR Supermix for Probes (no dUTP), 900 nM of PCR primers and 250 nM of probes for both target and reference regions plus 250 nM of unlabelled oligonucleotide and 0.5 U/μL of AluI restriction enzyme. A total of 5 ng of template genomic DNA was used per reaction. The droplet emulsions were prepared by mixing 70 μL of Droplet Generation oil for Probes with 20 μL of PCR reactions using the QX100 Droplet Generator (Bio-Rad). The PCR conditions were the following: for 5 min at 95°C, 40 cycles of 30 s at 94°C and for 1 min at 59.2°C for T-assay or 57.4°C for the C-assay, for 10 min at 10°C and a 4°C hold with ramp rate of 2°C/s. The fluorescence of each droplet was measured using a XQ200 Droplet Reader and QuantaSoft droplet reader software  was used to cluster droplets into distinct fluorescent groups. Copy number was determined by calculating the ratio of target (unknown) and reference concentration.The Bio-Rad QX 200 ddPCR system ,45 was uRBMY1 copy number is the sum of copy number estimates from the T- and C-assays.ddPCR reactions using the T-assay were performed at least twice for every sample. If the difference in copy number estimate between the two replicates was 0.8 or greater, then a third replicate was performed. The copy number estimates from the C-assay were consistently close to two for majority of the samples. Therefore, only samples that differed from two, plus 5% of all other samples, were replicated, each time giving a concordant copy number estimate. The total RBMY1 copy number and andrological parameters of 376 Estonian subjects was conducted using RStudio (version 1.1.456)  . Data we1.1.456) .RBMY1 copy number, calculated by the QuantaSoft software as the ratio of target and reference concentration (i.e. not binned or rounded), was used as a measure of real copy number in linear regression models. The following parameters were tested for association with RBMY1 copy number: sperm concentration (million/ml), total sperm count (million/ejaculate), semen volume (ml/ejaculate), total testis volume (ml), progressive motility (WHO motility classes A + B) (%), total testosterone (nmol/l), serum LH (IU/L) and serum FSH (IU/L). The genetic association tests were adjusted for appropriate cofactors; all parameters for age and additionally sperm parameters for abstinence time. For total testosterone level, an additional multiple linear regression analysis was conducted using both age and body mass index (BMI) as cofactors. For all linear regression analyses, natural log transformation was used to achieve an approximately normal distribution of values. Normality of the transformed distribution was assessed visually on histograms and statistically using the Kolmogorov–Smirnov test assuming that under the null hypothesis the observed values are normally distributed . In all cases, with the exception of total sperm count, the applied transformation resulted in a close-to-normal distribution.The RBMY1 copy number distribution difference in subjects with reduced (<32%) and subjects with normal (≥32%) progressive motility and Kruskal–Wallis test for the progressive motility distribution difference in samples with <10, 10 and >10 functional RBMY1 copies.The non-parametric Wilcoxon rank sum test was used to test for the P < 0.05 after adjustment for Bonferroni correction of multiple testing was considered as a statistically significant outcome.In all tests, HMG-2019-D-0019_Shi_Suppl_tables_ddz101Click here for additional data file.HMG-2019-D-0019_Shi_Supplementary_information_figures_ddz101Click here for additional data file.