Xist and then monitor the temporal changes in transcription and chromatin by allele-specific profiling. This reveals histone deacetylation and H2AK119 ubiquitination as the earliest chromatin alterations during XCI. We show that HDAC3 is pre-bound on the X chromosome and that, upon Xist coating, its activity is required for efficient gene silencing. We also reveal that first PRC1-associated H2AK119Ub and then PRC2-associated H3K27me3 accumulate initially at large intergenic domains that can then spread into genes only in the context of histone deacetylation and gene silencing. Our results reveal the hierarchy of chromatin events during the initiation of XCI and identify key roles for chromatin in the early steps of transcriptional silencing.During development, the precise relationships between transcription and chromatin modifications often remain unclear. We use the X chromosome inactivation (XCI) paradigm to explore the implication of chromatin changes in gene silencing. Using female mouse embryonic stem cells, we initiate XCI by inducing   •An epigenomic roadmap for initiation of X chromosome inactivation (XCI)•Histone deacetylation and H2A ubiquitination are among the earliest XCI events•HDAC3-mediated histone deacetylation is required for efficient XCI•PcG marks are first deposited intergenically and spread when gene silencing occurs Żylicz et al. provide a detailed characterization of the earliest stages of X chromosome inactivation, tracing chromatin modification dynamics and uncovering the key role of chromatin changes in initiation of gene silencing. Successful development requires the establishment and maintenance of euchromatin and heterochromatin in different parts of the genome. Progressively stable silencing of some genic regions occurs as epigenetic memory continues to accrue, finally leading to facultative heterochromatin formation . MultiplXist  have revealed that, upon Xist RNA coating, a program of striking chromatin rearrangements ensues. These include rapid loss of histone modifications associated with active promoters  and enhancers (H3K27ac and H3K4me1)  . FurtherH3K4me1) . These pH3K4me1) . The rea process ; howeverXist induction and elevated levels of heterogeneity in the population. These complications entirely mask primary chromatin events occurring in a small subset of cells embarking on the process of XCI. Recent advances in the identification of Xist binding proteins revealed that most histone-modifying activities are not directly recruited by Xist RNA but, rather, by its binding partners, like SPEN and HNRNPK  and differentiated cells, nothing is known about the dynamics of the XCI process, especially during early stages when gene silencing actually takes place . Specifid HNRNPK . A majorXist gene, we performed allele-specific native chromatin immunoprecipitation sequencing (ChIP-seq) as well as nascent transcript profiling at the initiation stages of XCI. Using 4-hr time resolution, we were able to reveal the precise order of chromosome-wide epigenetic events that are intricately coupled to gene repression during XCI. We find that loss of histone acetylation and, in particular H3K27ac, is one of the first events following Xist RNA accumulation during initiation of XCI. We also show that histone deacetylation, via HDAC3, is vital for efficient silencing of most genes on the Xist-coated X chromosome. Contrary to the prevailing hypothesis, HDAC3 is not acutely recruited to the X chromosome by Xist; rather, it is pre-loaded, mainly at putative enhancers. Upon Xist coating, pre-loaded HDAC3 likely mediates histone deacetylation and facilitates transcriptional silencing. We also uncover a surprisingly rapid accumulation of the PRC1-dependent H2AK119Ub mark on the X chromosome, particularly at intergenic regions lying in proximity to Xist RNA entry sites, which are pre-marked by Polycomb (PcG) marks. Accumulation of H3K27me3 appears slightly later and is delayed compared with gene silencing. Using Hdac3 and Xist mutant cells, we also show that spread of Polycomb marks into gene bodies can only occur when transcriptional repression begins. Our study reveals the chromatin choreography across the X chromosome during the early steps of XCI initiation and uncovers a role for histone deacetylation in the establishment of gene silencing.Here we investigate the earliest events accompanying transcriptional silencing of the X chromosome. Using a female ESC line in which one X chromosome can be specifically inactivated via an inducible Xist RNA coating, we used the TX1072 ESC line (Mus musculus castaneus x C57BL/6) line harbors a doxycycline (DOX) inducible promoter upstream of the endogenous Xist at the C57BL/6 (B6) allele ; this allelic information was then analyzed in detail to define XCI-specific changes.A molecular roadmap of the earliest chromatin changes during the initiation of XCI has not yet been established. To reduce heterogeneity and obtain a highly resolved time series of chromatin events upon ESC line . This hy) allele . By addi changes C. We per changes A and S1B changes B and S1EXist coating, the Xi becomes rapidly depleted of active histone modifications. Although this global loss of euchromatin occurs in a similar time window as the changes in X-linked transcriptional activity . This parameter of allelic skewing uses the active X chromosome as a powerful internal control, rendering the analysis less sensitive to variability in nChIP-seq quality and, thus, more stable between replicates.Upon activity , the preactivity E. This r of loss D. To ana of loss . For eacXist induction  as a function of time and fitted a sigmoidal curve . We founpromoter D. Similapromoter C. This r element I. Thus, 50; 50 analysis revealed that both H3K27ac and H3K4me3 become depleted concomitantly , which probably reflects an intermediate of stepwise enzymatic H3K4me3 demethylation , we first tested selective pharmacological inhibitors against all HDACs expressed in ESCs A. Importgulation B. The fi factors . Also, Hne: Gpc4 .Figure SHdac3−/− TX1072 deletion clones using CRISPR/Cas9 technology  in their silencing by the HDAC3 loss and three less affected genes   E.Hdac3−/− ESCs with selective inhibitors against all expressed HDACs . We found that allelic skewing  of AtrX, G6pdX, and Rnf12 were not affected in any of the treated cells  expressed nearly normal levels of HDAC3   C. Among licates) . Intrigupartners , only RYpartners C. Our reXist locus and at some HDAC3 peaks  F. The vaC3 peaks D and S5Eas found F. On theas found G. In facas found H. Finallas found I. In sumXist RNA coating, in particular H2AK119Ub (PRC1-dependent) and H3K27me3 (PRC2-dependent). However, the early dynamics of this process, as well as the relative timing of H3K27me3 and H2AK119Ub deposition, have remained largely unexplored. To address this question, we have performed nChIP-seq for both PcG-dependent marks. These datasets showed a reproducible and stable pattern of enrichment on autosomes, both between time points and biological replicates  as well as the first regions with which it associates   . There i50 analysis revealed that H2AK119Ub accumulates first intergenically and is deposited only later at active gene bodies, promoters, and putative enhancers (Rnf12) or slowly (Huwe1 and Alg13) or escape the process altogether (Kdm6a) (50 parameter for H2AK119Ub (We next investigated the accumulation dynamics of both marks at specific types of genomic windows. EDnhancers F. This pnhancers E. This i (Kdm6a) F and 6G.2AK119Ub G. This i2AK119Ub I. This r2AK119Ub H. Thus, Kdm6a; We also investigated genes that normally escape from XCI (constitutive) or escape more variably and in specific tissues (facultative escapees) . We examXist RNA interacts  and that correspond to some of the first regions with which nteracts . We demoXist mutant lacking the A-repeat element (Xist:ΔA). Xist:ΔA RNA is able to coat the X chromosome and lead to PcG mark accumulation (based on IF) but cannot initiate gene silencing  or mutant Xist (TXY:XistΔA) under a DOX-inducible promoter at the endogenous locus  to transcribed loci gain normal levels of H3K27me3 and intermediate levels of H2AK119Ub, as exemplified in genome browser tracks  accumulate neither H2AK119Ub nor H3K27me3 (Hdac3−/− (resistant) robustly accumulated both marks. These relationships are exemplified by a AtrX (resistant gene) and Rnf12 (sensitive gene)  G. Thus, Xist spreading or the presence of transcription , lying in 3D proximity to the Xist locus Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Edith Heard (Mus musculus castaneus X C57BL/6] embryonic stem cells) cells have been previously derived in the lab (Hdac3−/− and Hdac3Flag/Flag clones have been derived from the TX1072 line by CRISPR/Cas9 targeting. Cells were cultured on gelatine-coated plates in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 15% fetal bovine serum, 2-mercaptoethanol (0.1mM), LIF(1000u/mL) and 2i  . TXY:Xist and TXY:XistΔA  were obtained from the Wutz team . Cells were then collected at regular intervals. For TXY based ESC lines we have plated them at a density of 1mln/T75. After 24hrs of culture in DMEM/15%FCS+LIF cells were washed twice with PBS and medium was changed to DMEM/10%FCS-LIF with or without doxycycline (1.5ug/ml). Cells were collected after 48hrs of differentiation. For HDAC inhibition cells were pre-treated for 1hr with: 10 μM BRD668; 10 μM RGFP966; 10 μM Tasquinimod; 5 μM Tubastatin A; 5 μM PCI-34051; 30nM LMK235.Xist induction. Cell pellet was resuspended in 90 μL (per 10 mln cells) of Lysis Buffer . After lysing cells on ice for 10 min we added 62 μL (per 10 mln cells) of Lysis Buffer with MNase (500 μL buffer + 0.5 μL MNase). Chromatin was digested for exactly 10 min at 37°C and reaction was stopped by the addition of 20 mM EGTA. To remove undigested debris the lysates were centrifuged at 13000 rpm for 5 min at 4°C. Supernatant was transferred to a fresh tube, an equal volume of STOP Buffer  was added, samples were stored on ice.Cells were collected using Accutase (Thermo Fisher Scientific), washed twice in ice-cold PBS and counted. Typically, 3.5mln cells were used per immunoprecipitation (IP). A fraction of cells was always used for RNA/FISH verification of 5 μL of lysate was digested in 45 μL of ProtK Digestion Buffer  for 30 min at 56°C. 50 μL of AMPure XP beads were added to the digested lysate together with 60 μL of 20% PEG8000 1.25M NaCl. After mixing the samples were incubated for 15 min at RT. Beads were separated on a magnet and washed twice with 80% Ethanol for 30sec. DNA was eluted in 12 μL of Low-EDTA TE and DNA concentration was measured using Qubit DNA High-Sensitivity kit. These measurements were used to normalize lysate concentration between samples. DNA isolated in this step was used as the input sample. The volume of each undigested lysate was adjusted for equal concentration and to obtain 1mL per IP using a 1:1 mix of Lysis Buffer and STOP Buffer.Anti-mouse Dynabeads (50ul/IP) and Protein-A Dynabeads (10ul/IP) were washed twice in Blocking Buffer . Beads were then resuspended in Blocking buffer and coated with antibodies for 4hrs at 4°C . Once coated beads were magnet-separated and resuspended in 1mL of concentration-adjusted lysate. Samples were left rotating overnight at 4°C.Following day beads were magnet-separated and washed quickly with ice-cold washing buffers. anti-H3K4me3 IP was washed 8 times with Low Salt Buffer . All remaining IPs were washed 4-times with Low Salt Buffer, 2-times with High Salt Buffer  and 2-times with LiCl buffer . Prior to elution all samples were rinsed once in TE. ChIP-DNA was eluted in ProtK-Digestion buffer for 15 min at 56°C. Beads were separated and the supernatant was further digested for another 2 hr at 56°C. DNA was isolated using AMPure XP beads as described for the input sample.® Ultralow Library System V2 following suppliers protocol. Amplified libraries were size-selected for dinucleotide fraction (350-600bp fragments) using agarose gel-separation and MinElute Gel Extraction Kit (QIAGEN). Sample quality was inspected using D1000 tapestation. Samples were sequenced with HiSeq2500 using PE100 mode for nChIP-seq on TX1072 cell line and SE50 mode for nChIP-seq on TXY cell line.For each nChIP-seq, 0.5 μL of each sample was used for qPCR validation of enrichment at control regions. 0.5 μL of input samples were also used to verify the digestion efficiency using D1000 tapestation. Remaining DNA concentration was adjusted and used for library preparation using Ovation5 of ESC were plated onto fibronectin-coated 6-well in N2B27 medium  with bFGF (12 ng/ml) and ActivinA (20 ng/ml) and with or without doxycycline (1ug/mL). Medium was changed every day and cells were collected at appropriate intervals.2.5x10Huwe1 and Atrx, a BAC spanning the respective genomic region  was labeled by nick translation (Roche) using spectrum green (Abbot). Per coverslip, 60ng probe was ethanol precipitated with Cot1 repeats, resuspended in formamide, denatured (10min 75°C) and competed for 45min at 37°C. For Xist detection or intron-spanning plasmid probe p510 was labeled by nick translation (Roche) using spectrum red (Abbot). Probe was ethanol precipitated and resuspended in formamide and denatured (10min 75oC). Both Xist and AtrX or Huwe1 probes were co- hybridized in FISH hybrization buffer  over-night. Washes were carried out at 42°C three times 5min in 50% formamide in 2X SSC at pH = 7.2 and three times 5min in 2X SSC. 0.2mg/ml DAPI was used for counterstaining and mounting medium consisted of Vectashield (Vectorlabs). Images were acquired using an Inverted Confocal Spinning Disk Roper/Nikon.ESC were dissociated using Accutase (Invitrogen) and adsorbed onto Poly-L-Lysine (Sigma) coated coverslips #1.5 (1mm) for 5 min. Cells were fixed with 3% paraformaldehyde in PBS for 10 min at room temperature and permeabilised for 5 min on ice in PBS containing 0.5%Triton X-100 and 2mM Vanadyl- ribonucleoside complex (New England Biolabs). Coverslips were preserved in 70% EtOH at −20°C. Prior to FISH, samples were dehydrated through an ethanol series  and air-dried quickly. For detecting Xist detection. Here we used a custom designed strand-specific probe that covers all exons with ∼75 bp long oligo nucleotides end-labeled with the Alexa 488 fluorophore (Roche). Probes was hybridized in FISH hybridization buffer  over-night. Washes were carried out at 42°C three times 5min in 50% formamide in 2X SSC at pH = 7.2 and three times 5min in 2X SSC. 0.2mg/ml DAPI was used for counterstaining and mounting medium consisted of Vectashield (Vectorlabs). Images were acquired using an Inverted Confocal Spinning Disk Roper/Nikon. For signal quantification, signal intensity was measured along an axis across the Xist domain (z-plane with Xist maximum intensity) and part of the nucleus. Signal was not measured across the nucleoli. Profiles from 70-100 nuclei were centered at the point of Xist cloud boundary . Signal was normalized at per cell basis. In Cells were grown on fibronectin coated coverslips #1.5 (1mm) and treated with doxycycline (1ug/mL) for 24hrs. Cells were fixed with 3% paraformaldehyde in PBS for 10 min at room temperature and permeabilised for 5 min on ice in PBS containing 0.5%Triton X-100 and 2mM Vanadyl- ribonucleoside complex (New England Biolabs). Samples were blocked for 15 min at room temperature with 1%BSA/PBS. Coverslips were incubated with primary antibodies diluted in blocking solution  in the presence of a Ribonuclease Inhibitor  for 45 min at room temperature. Coverslips were washed thrice with PBS for 5min and subsequently incubated with secondary antibody solution  for 45 min at room temperature. Coverslips were washed three times with PBS for 5 min at room temperature. Cells were fixed again with 2% paraformaldehyde in PBS for 10 min at room temperature. After rinsing twice in 2xSSC coverslips were hybridized for RNA was extracted according to the manufacturer’s recommendations using RNeasy Mini Kit (QIAGEN) with on-column DNase digestion (QIAGEN). For cDNA synthesis 1.1 μg RNA was reverse transcribed using Superscript III Reverse Transcriptase (Thermo Fisher Scientific). For allelic-skewing analysis the cDNA was PCR-amplified with biotinylated primers and sequenced using the Pyromark Q24 system (QIAGEN). To quantify ChIP DNA enrichments at control regions 2x SybRGreen Master Mix (Applied Biosystems) and a ViiA7 system (Applied biosystems) were used.RNA was prepared as described above and quality of samples were verified by Tapestation. Only samples with RIN score above 9 were processed. 100 ng of RNA was used for library preparation using Ovation Mouse RNA-Seq System that uses mouse-specific InDA-C depletion of rRNA. Manufacturers recommendations were followed. Libraries were sequenced using HiSeq2500 at PE100 settings.The TT-seq protocol was performed as previously described with modifications . Cells win vitro with MEGAscript T7 Transcription Kit (Thermo Fisher), where 1/10 UTP in the transcription reactions was replaced with 4sUTP (Jena BioScience) for the 4sU-labeled spike-ins. RNA lysates were thawed on ice and 24 ng of spike-in mix was added per 10 million cells. The RNA was then purified according to the manufacturer’s protocol. 4sU-labeled RNA was isolated from 300 μg total RNA, for this two aliquots of 150 μg were sheared in Covaris MicroTubes with the Covaris S220 System (Covaris) for 10 s with settings 100 W and 1% duty cycle. These were then pooled and 2 μg of sonicated total RNA was taken for total RNA fraction. The RNA was biotinylated with EZ-Link HPDP-Biotin (Thermo Fisher) in two biotinylation reactions per sample  that were incubated 1.5 h at 24°C. The biotinylated RNA was extracted with chloroform and precipitated with isopropanol. 4sU-labeled RNA was isolated by incubating the biotinylated RNA with paramagnetic streptavidin μMACS MicroBeads (Miltenyi) for 15 min at room temperature and binding to MACS columns. The columns were washed three times with wash buffer  at 65°C and three times with wash buffer at room temperature. The RNA was eluted from the columns with 100 mM DTT. The eluted 4sU-labeled RNA and reserved total RNA were purified with the miRNeasy Micro kit (QIAGEN) according to the manufacturer’s protocol with on-column DNase I treatment. 150 ng and 500 ng of isolated newly synthesized RNA and total RNA fractions, respectively, were used to prepare NGS libraries with the TruSeq Stranded Total RNA Library Prep Kit (Illumina). The sample libraries were pooled and sequenced as a single run on the Nextseq550 (Illumina) using PE150 mode.The spike-in mix, containing three 4sU-labeled and three unlabelled RNAs, was synthesized Hdac3−/−#1 harbors a deletion of exon 4-7 on the Cast allele and exon 9 on the B6 allele. Hdac3−/−#2 harbors a deletion of exon 4-7 on the B6 allele and exon 7 on the B6 allele. Both mutants showed negligible expression on the mRNA level as well as no detectable protein when using a N terminus specific antibody  were PCR amplified from mouse genomic DNA. 1-step Gibson cloning (NEB) was subsequently used to simultaneously surround the digested AID-3xFLAG insert (carrying a puromycin resistance gene under the control of the PGK promoter) in frame with the homology arms and clone the insert into the pBR322 vector. Synonymous mutations in the PAM/SEED target sequence (located on the 5′ homology arm) were then introduced using the QuickChange II XL site-directed mutagenesis kit (Agilent) to prevent Cas9 mediated cleavage of the targeting vector upon transfection and of the 3xFLAG tagged allele(s) upon integration.The Single gRNAs has been designed as previously described  and clonHdac3 locus was validated by sanger sequencing and western blotting and kept for subsequent experiments  were trypsinised and washed in PBS. Pellets were snap frozen and stored at −80°C. Defrosted pellets were resuspended in 10 mL of buffer A  and incubated for 10 min at 4°C. Nuclei were pelleted at 2000 rpm at 4°C for 10 minutes. Nuclei were resuspended in 500μL of buffer C  glycerol; 150 mM KCl; 5 mM MgCl2; 2 mM EDTA; 2 mM EDTA; 1 mM DTT; protease inhibitors) and sonicated 3 times for 10 s . Lysates were cleared by centrifugation 12000 rpm at 4°C for 20 minutes. Lysate concentrations were equalised based on protein concentration. Anti-FLAG M2 Magnetic Beads (Sigma-Aldrich) were washed twice in buffer C and then added to each lysate (50μL per IP). Lysates were left rotating over-night at 4°C. Next day beads were washed 5 times in wash buffer .50 mln (per IP) ESCs  for 1 hour in 100μL of 25mM NH4HCO3. Samples were then loaded onto a custom-made C18 StageTips for desalting. Peptides were eluted using 40/60 MeCN/H2O + 0.1% formic acid and vacuum concentrated to dryness.Online chromatography was performed with an RSLCnano system  coupled online to an Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientific). Peptides were trapped on a C18 column  with buffer A (2/98 MeCN/H2O in 0.1% formic acid) at a flow rate of 4.0μL/min over 4 min. Separation was performed on a 50cm x 75 μm C18 column  regulated to a temperature of 55°C with a linear gradient of 5% to 25% buffer B (100% MeCN in 0.1% formic acid) at a flow rate of 300 nL/min over 100 min. Full-scan MS was acquired in the Orbitrap analyzer with a resolution set to 120,000 and ions from each full scan were HCD fragmented and analyzed in the linear ion trap.Cells were grown in 145mm plate format. Cells were initially fixed in 1.5 mM ethylene glycolbis [succinimidyl succinate] (EGS) for 30 min, next 2% formaldehyde was added and further incubated for 10 min. After rinsing twice in PBS, cells were scraped and pellets directly used for ChIP.Fixed pellets were lysed in 6 mL Nuclear Lysis Buffer  for 10min on ice and dounce-homogenized. Nuclei were pelleted and resuspended in 0.9 mL Lysis Buffer . Chromatin was sheared using a BioruptorPlus (Diagenode) set to high for 30 cycles. Unsonicated chromatin was removed by centrifugation. Supernatant was diluted with 3.6 mL of Dilution Buffer  and antibody coated beads were added (per IP: 10 μL of anti-FLAG antibody). Samples were left rotating overnight at 4°C.Following day beads were magnet-separated and washed 5-times with LiCl buffer  and 5 times with High Salt Buffer . Each wash was performed for 10 min on a rotating wheel at 4°C. Prior to elution all samples were rinsed once in TE. ChIP-DNA was eluted in ProtK-Digestion Buffer  for 15min at 56°C. Beads were separated and the supernatant was further digested for another 2hrs at 56°C and then decrosslinked for 4hrs at 68°C. DNA was isolated using AMPure XP beads. Libraries were prepared using Ovation Ultralow Library System V2 following suppliers protocol. Samples were sequenced with HiSeq2500 using PE100 mode.http://www.bioinformatics.babraham.ac.uk/projects/trim_galore) and Cutadapt (1.8.2)  with options [--end-to-end -N1 -q] (https://broadinstitute.github.io/picard/) and reads mapped on blacklisted regions from Encode Consortium were discarded. SNPSplit (0.3.2)  have been removed with TrimGalore  . An ‘N-m (1.8.2)  which is -N1 -q] . Duplica (0.3.2)  was then (0.3.2) , using aPeak calling was done with MACS2 (2.0.10)  with optB6/(readsCast+ readsB6)], were calculated.Total and allelic reads overlapping consensus peaks were counted using featureCounts (1.5.1), with options [ -C -p -P] . Only pe50, which corresponds to the maximum slope of the sigmoidal fitting; and the minimum second derivative used as a proxy for when the curve starts to decrease. The minimum of the second derivative has been calculated using optimize function from R in the interval between 0 and the calculated ED50 to avoid finding a local minimum not corresponding to the real minimum. IC/ED/minimum second derivative superior to 24h (outside the time range of the experiment) or not calculated were considered windows with a late loss of active histone mark, and replaced by 24h.The dynamic evolution of d-score with time was analyzed for each peak with a d-score calculated on the 10 samples . Moreover, only peaks that had a biallelic d-score at time 0 (comprised between 0.3 and 0.7) were selected. Then normalized d-scores were calculated by dividing each d-score by the corresponding initial d-score (time 0) and by 2, so that initial normalized d-scores start at 0.5. Initially we have fitted either exponential or sigmoidal curve to these values. We compared the quality of this fitting by taking into account the number of parameters used in the models (AIC and BIC criteria) and concluded that the sigmoidal model best reflects the observed dynamics (data not shown). Sigmoidal fitting of normalized d-scores in function of time has been done for each peak with a four-parameter log-logistic function from drc R package . SigmoidMoreover, for all active histone marks, IC25, IC30 and IC40  were also tested and were able to replicate all our major findings (data not shown).Xist, distance to the closest TAD boundary , distance to boundary , distancboundary , LINE dery sites (< 100kb)For comparison of dynamics E of deacAverage plots of H3K4me1 signal were created using DeepTools (3.0.2) . Matrix For repressive marks, global analysis was first done on fixed windows (10 kb) spanning the whole genome, then on different genomic subcategories: active gene bodies, active promoters, active enhancers and intergenic regions. Active genes were defined as genes with a transcript having its TSS (refFlat annotation) overlapping a consensus peak of H3K9ac and a consensus peak of H3K4me3. For genes having several active transcripts detected, the active gene was defined as starting at the minimum start of transcripts, and ending at the maximum end of transcripts. Then, the active gene bodies were defined as those active genes excluding the 2 first kb downstream of TSS. Active promoters were defined as ± 2kb windows around the TSS of active genes. Putative, active enhancers were defined as intersect between consensus H3K27ac peaks and consensus H3K4me1 peaks, located at a minimal distance of 1kb from a TSS. Intergenic regions were defined as 10 kb windows not overlapping a gene (active or inactive) and its promoter (2kb downstream) or an active enhancer. Enhancer coordinates were also used for the analysis of active histone mark (H3K4me1 and H3K27ac).It should be noted that a d-score analysis, as used for active mark loss, involves the use of the active allele as an internal control, but in the case of H2AK119Ub and H3K27me3, this was hindered by very low enrichment for repressive marks across the Xa. For all defined windows, total and allelic reads overlapping those features were then counted using featureCounts (1.5.1), with options [-C -p -P] . Then, a50 and the maximum second derivative of the sigmoidal fitting were calculated for each window. Windows with ED50/maximum 2nd derivative superior to 24h, or not calculated, were considered as windows with a late accumulation of repressive marks, those were then replaced by 24h. For pairwise comparison between marks, only windows with ED50/maximum 2nd derivative inferior to 24h for both marks were selected. For comparison of ED50 between different genomic subcategories  for a same histone mark, no filter was done.Sigmoidal fitting of B6 read accumulation in function of time has also been done with the four-parameter log-logistic function from drc R package. Sigmoidal fittings with low residuals  + 1.5 sd) were selected, representing more than 90% of the analyzed windows. The ED50, IC60 and IC65 as criteria. All those analysis revealed stable and reproducible results (data not shown).To test the stability of our results, main analysis was also performed with different thresholds of residuals . Moreover, repressive marks dynamics were also analyzed using allelic ratio (d-scores), such as for analysis of active histone marks, using EDMus musculus CAST/EiJ and Mus musculus C57BL/6) with STAR 2.3.0  and low quality bases (< Q20) have been removed with Cutadapt (v1.16) . For allAR 2.3.0  with maxAR 2.3.0  was usedAR 2.3.0  was usedAR 2.3.0  on longeReads were first mapped on rRNA with Tophat (2.1.0) , with opFor differential gene expression analysis prior to DOX treatment, analysis was done on genes for which 2 among the 4 samples have a TPM superior to 1. Normalization was done using TMM  method fB6/(readsB6 + readsCAST)) / (1-(readsB6/(readsB6 + readsCAST))).The processing from raw data (fastq) to clean mapped file was done such as for time course nChIP-seq. Then, for active histone marks, the reads counts of H3K27ac and H4ac nChIP-seq were done on consensus H3K27ac peaks and consensus H4ac peaks defined in time course analysis, using featureCounts (1.5.1), with options [-C -p -P] . D-score∗ d-score). After this adjustment, only the peaks that were biallelic at time 0 in WT and mutant samples were selected (d-scores between 0.3 and 0.7) for analysis. For repressive marks, analysis was done such as for time course nChIP-seq, based on signal accumulation of 10 kb windows all along the X chromosome.Then d-score for this mutant was adjusted from classic d-score as follow: d-score = d-score/ for both clones  at each time point were selected. D-scores were normalized to time 0 (d-score / d-score t0), then only peaks/genes considered as not escaping were selected . Difference of normalized d-score at 24h between WT and mutant was then calculated, and divided by the difference of normalized WT d-score between 0 and 24h for the WT sample. K-means with 3 clusters was done on this normalized difference, identifying 3 groups: sensitive, intermediate and resistant peaks/genes. For nChIP-seq on repressive histone marks, differential enrichment between WT and mutant was defined as the difference between WT normalized BL6 enrichment relative to time 0 and Mutant normalized BL6 enrichment relative to time 0, divided by WT normalized BL6 enrichment relative to time 0.The processing from raw data (fastq) to d-score calculation was done such as for time course nChIP-seq, with slight modifications. The peak calling was done with MACS2 (2.0.10)  with optHdac3Flag/Flag ESC cell line using DeepTools bamCompare with options [--ratio subtract --binSize 100] before to proceed to the next step. Then, matrix counts were created using DeepTools computeMatrix around TSS of active promoters (with option [--binSize 100]) or centers of putative enhancers, plots were then created using DeepTools PlotHeatmap.Heatmaps and average plots of HDAC3 signal in comparison with H3K27ac and H4ac signal were created using DeepTools (3.0.2) . Merged For H3K27ac and HDAC3 peaks annotation comparison C, peaks Category of peaks accumulating signal or not on Xi E and S5FProcessing steps to clean bam files were similar to time course nChIP-seq analysis, except that mapping was not allele-specific but done on mm10 genome and that reads mapped with low quality (< q10) were removed with samtools (1.3). Such asThe windows for repressive mark analysis were defined similarly as for nChIP-seq time course analysis for repressive marks, except that active TSS were defined here based on consensus peaks of H3K27ac overlapping TSS, and putative active enhancers as H3K27ac peaks located at minimum 1kb of TSS.Reads overlapping defined peaks (for active marks) or windows (for repressive marks) were then counted with featureCounts (1.5.1)  with defFor global analysis on repressive marks, counts normalization was done such as for time course nChIP-seq analysis, based on counts falling in autosomal consensus peaks. For windows and peaks analysis, windows that had less than one read per 50kb for more than 2 among the 8 samples were removed for the analysis. Normalization factors were calculated based on windows located on autosomes , with TMM method using edgeR .Xist Cloud by FISH experiments , linear regression including the percentage of induction calculated by FISH was fitted for each window according the following model: ∼0 + clones + clones: induction, using Voom function from Limma R package (Because of the high variability in proportion of cells involved in X chromosome inactivation quantified by the presence of  package . The sloMetaplots were created using DeepTools (3.0.2) . BigwigsMus musculus (UP000000589) UniProt database using Sequest HF through proteome discoverer (version 2.2). Enzyme specificity was set to trypsin and a maximum of two-missed cleavage sites were allowed. Oxidized methionine, N-terminal acetylation, and carbamidomethyl cysteine were set as variable modifications. Maximum allowed mass deviation was set to 10 ppm for monoisotopic precursor ions and 0.6 Da for MS/MS peaks.For identification, the data were searched against the p-values were adjusted with a Benjamini–Hochberg FDR procedure with a control threshold set to 0.05.The resulting files were further processed using myProMS v3.6 . FDR calGSE116480. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD011344The accession number for the sequencing datasets reported in this paper is GEO:


The TiO2 nanorods were characterized using X-ray diffraction (XRD), energy dispersive X-ray spectrometry (EDX), transmission electron microscopy (TEM), scanning electron microscopy (SEM), and optical contact angle testing. The results showed that the nanorods with diameters of 100–500 nm and lengths of 100 nm to 1 μm were obtained on the Ti foil surface. The length and density of the TiO2 nanorods were perfect at the conditions of HCl concentration 0.5 mol/L, temperature 220 °C, and reaction time 12 h. The TiO2 nanorods formed parallel to the consumption of Ti and grew along the (110) direction having a tetragonal rutile crystal. The morphology of the nanorods possessed a three-dimensional structure. The contact angle of the nanorods was only 13 ± 3.1°. Meanwhile, the photocatalytic activities of the TiO2 nanorods were carried out using ultraviolet fluorescence spectrophotometry for the methyl orange detection, and the degradation was found to be about 71.00% ± 2.43%. Thus, TiO2 nanorods can be developed by a one-step acidic hydrothermal method using Ti foil simultaneously as the substrate with a TiO2 source; the TiO2 nanorods exhibited photocatalytic performance while being environment-friendly.Three-dimensional and dendritic rutile TiO When the HCl concentration was 0.5 mol/L, reaction temperature 220 °C, and reaction 12 h, the morphology and density of nanorods reached an optimum with hydrophilic and photocatalytic properties. The hydrophilic and photocatalytic activity of TiO2 nanorods reached a maximum, when the contact angle was only 13 ± 3.1° and the degradation 71.00% ± 2.43%, respectively. It can be concluded that TiO2 nanorods can be developed by a one-step acidic hydrothermal method using Ti foil simultaneously as the substrate and Ti source, and the TiO2 nanorods thus formed exhibit photocatalytic performance while being environment-friendly.In summary, three-dimensional dendritic rutile TiO


JARID2 is a non-catalytic member of the polycomb repressive complex 2 (PRC2), which is known to regulate developmental target genes in embryonic stem cells. Here, we provide mechanistic insight into the modulation of Wnt signaling by JARID2 during murine skeletal muscle differentiation.me3) were observed on both promoters. We found that activation of the Wnt signaling pathway upregulated MYOD and restored differentiation. Activation of the Wnt pathway in JARID2 depleted cells caused β-catenin to translocate to the nucleus, where it bound to and activated the Myod1 promoter. We show that the Wnt antagonist SFRP1 is highly upregulated in the absence of JARID2 and is a direct target of JARID2 and the PRC2 complex. Ectopic expression of SFRP1 blocked MYOD and late muscle gene expression and inhibited the translocation of β-catenin to the nucleus. Finally, we show that JARID2 and SFRP1 are inversely correlated in melanoma, confirming that the JARID2-mediated repression of SFRP1 extends beyond skeletal muscle and has important implications in many cellular systems, including cancer.We show that JARID2 is expressed in proliferating myoblasts, but downregulated upon muscle differentiation. Unexpectedly, depletion of JARID2 or the catalytic subunit of the PRC2 complex, EZH2, inhibited differentiation, suggesting that JARID2 and the PRC2 complex are required to initiate this process. Expression of the myogenic regulatory factors required to promote differentiation, MYOD and MYOG, was downregulated in the absence of JARID2, even though decreases in the methylation of histone H3 lysine 27  contains supplementary material, which is available to authorized users. Myod1  me3 , anti-pan methyl H3K9  and anti-β-catenin . Rabbit IgG (SCBT) was used as a nonspecific control. Primers are described in Additional file ChIP assays were performed as described previously . The folhttp://cancergenome.nih.gov/, as described [JARID2 and SFRP1. The expression data downloaded from TCGA for all the patients were used for Pearson’s correlation test between JARID2 and SFRP1, and scatterplots were generated. Student’s t test was performed between non-overlapping high- and low-group patients for JARID2 and SFRP1 expression level and was plotted as box plots. All the statistical analysis and graphs were made using SPSS Statistic 24.0.0 software.The “OncoLnc” tool was used to download patient information on skin cutaneous melanoma (SKCM) from The Cancer Genome Atlas (TCGA) database: escribed . Using Kt tests or one-way ANOVA followed by Tukey’s HSD (honest significant difference) post hoc test. For one-way ANOVA analysis, all means not sharing the same letter were statistically significant. Probability value of < 0.05 was taken to indicate significance.Data are presented as means ± standard errors (S.E.). Statistical comparisons were performed using unpaired two-tailed Student’s Additional file 1: Figure S1. Myod1 and Myog are direct targets of the PRC2 complex. a–cMyod1 is a direct target of JARID2 and the PRC2 complex. ChIP assays using antibodies against trimethylation of lysine 27 of histone 3 (H3K27me3) and a nonspecific antibody (IgG) were performed on C2C12 cells stably expressing scr and shJarid2. Primers spanning three regulatory regions of the Myod1 promoter were used: core enhancer (CE) (a), distal regulatory region(DRR) (b) and proximal regulatory region (PRR) (c). d, e. Myogenin is a direct target of JARID2 and the PRC2 complex. Cells from (a) were differentiated for 2 days and subjected to ChIP assays performed and analyzed as in (a). Primers spanning two different regions of the myogenin promoter, 1.5-kb upstream of the transcription start site (1.5 kb) (d) and proximal promoter  (e) were used. n.s. is not statistically significant. F. H3K27me3 at the HoxB7 promoter is unaffected by depletion of JARID2. ChIP assays were performed and analyzed as in E.G. H3K9 methylation of Myod1 is not dependent on JARID2. ChIP assays using antibodies against trimethylation of lysine 9 of histone 3 (H3K9me) and a nonspecific antibody (IgG) were performed on C2C12 cells stably expressing scr and shJarid2. Primers spanning three regulatory regions of the Myod1 promoter were used: core enhancer (CE) (g), distal regulatory region (DRR) (h), and proximal regulatory region (PRR) (i). j H3K9 methylation is not observed on the Myog proximal promoter. ChIP assays were performed as in g. k H3K9 methylation is reduced on the upstream 1.5-kb region of Myog when JARID2 is depleted. ChIP assays preformed as in G. Error bars are S.E.M. **p value < 0.01 and ***p value < 0.001. n ≥ 4.Additional file 2: Figure S2. Exogenous expression of MYOD restores myogenin and late muscle gene expression in JARID2 depleted C2C12 cells. A. Plasmids expressing Myod1 (cDNA) or empty vector were transiently transfected in C2C12 cells stably expressing shRNA against Jarid2 mRNA. Total RNA was extracted 48 h post-transfection (UD) or 96 h post-transfection with 48 h in low serum media (D2). mRNA expression of Myod1 was assayed by qRT-PCR. Relative mRNA expression was calculated relative to the vector UD sample. Error bars are S.E.M. ***p value < 0.001 versus time matched vector. n ≥ 3. b Myogenin expression is restored upon exogenous expression of MYOD. mRNA expression of myogenin was analyzed by qRT-PCR as in a. c Late muscle gene expression is induced upon transient MYOD expression. Cells from (a) were used to assay for mRNA expression of myosin light chain (Mylpf) by qRT-PCR and analyzed. Error bars are S.E.M. ***p value < 0.001 versus time-matched vector. n ≥ 3.Additional file 3: Figure S3. Expression of GSK3β is unaltered in Jarid2 depletions irrespective of Wnt activation. C2C12 cells stably expressing scramble shRNA (scr) or Jarid2 mRNA-specific shRNA (shJarid2) were grown to confluency in high serum (UD) and switched to differentiation conditions for 2 days (D2). shJarid2 cells were treated with either 10 mM NaCl or 10 mM LiCl for 2 days in differentiation conditions as indicated. Total cell extracts were probed as indicated. Gels were quantified and normalized to respective loading controls (lower panel). Relative expression was calculated relative to scr UD sample and plotted as bar graphs. Error bars are S.E.M. n ≥ 3.Additional file 4: Figure S4. Nkd1 is not activated by JARID2 depletion. aNkd1 mRNA is downregulated in JARID2 depleted cells as assayed by qRT-PCR. Error bars are S.E.M. **p < 0.001 versus scr. n ≥ 3. b The Nkd1 promoter is methylated in a JARID2 dependent manner, scr or shJarid2 cells were used for ChIP assays using antibodies against trimethylation of histone 3 lysine 27 (H3 K27me3) and nonspecific antibody (IgG) with primers specific to the Nkd1 promoter. Error bars are S.E.M. n ≥ 3.Additional file 5: Figure S5. Expression of JARID2 and SFRP1 is better correlated in groups divided based on JARID2 expression. a, b Box plot representing the expression of JARID2 mRNA (a) and SFRP1mRNA (b) in between lower 25 percentile and upper 25th percentile, respectively (p < 0.001). c Pearson’s correlation test was performed between the expression of JARID2 mRNA and SFRP1 mRNA after splitting all patients into two categories, top 25 percentile and bottom 25 percentile, based on expression of JARID2 mRNA.Additional file 6: File S1. TCGA data analysis used in study.Additional file 7: Table S1. Oligonucleotides used in study.


Phage display is a powerful technique for drug discovery in biomedical research in particular for antibody libraries. But, several technical challenges are associated with the selection process. For instance, during the panning step, the successful elution of the phages bound to the antigen is critical in order to avoid losing the most promising binders. Here, we present an efficient protocol to establish, screen and select synthetic libraries of domain antibodies using phage display. We do not only present suitable solutions to the above-mentioned challenges to improve elution by 50-fold, but we also present a step by step in-depth protocol with miniaturized volumes and optimized procedures to save material, costs and time for a successful phage display with domain antibodies. Hence, this protocol improves the selection process for an efficient handling process. The here presented library is based on the variable domain (vNAR) of the naturally occurring novel antibody receptor (IgNAR) from cartilage fishes. Diversity was introduced in the Complementarity-Determining Region 3 (CDR3) of the antigen-binding site with different composition and length.  Antibodies in particular immunoglobulin G (IgGs) are some of the most important biopharmaceutical molecules with a highly relevant market volume. Antibodies or other scaffolds have broad and diverse applications for the therapy of several diseases since they can bind almost any pharmaceutical target. To develop suitable therapeutic antibodies an efficient screening and selection technology is phage display, which allows the screening of large libraries ,2.Antibody phage libraries have become practical tools for the generation of monoclonal antibodies (mAb), antibody fragments like single-chain variable fragment (ScFv), antigen-binding fragments (Fab) or recombinant single-domain antibodies from camel (VHH) or shark (vNAR). The success of the selection process depends on a variety of elements including the quality and diversity of the initial library. The initial library can be either of a fully synthetic, semi-synthetic, naïve (non-immunized) or immunized (antigen-specific) origin ,4,5,6. HE. coli (Escherichia coli) when phage display will be used as a technique for screening. The variable domain of shark antibody has four (hyper) variable loops: CDR1, hypervariable loop 2 (HV2), hypervariable loop 4 (HV4) and CDR3. Several studies have shown that the diversity of antibodies belongs predominantly to CDR3 loops which are tolerant to sequence and length variations  supE thi-1 Δ(lac-proAB) Δ(mcrB-hsdSM)5, (rK-mK-) .Phagemid pSEX81 Hyperphage Oligonucleotide primers for PCR are listed in ®/MicroPulser™ Electroporation Cuvettes, 0.2 cm gap Gene PulserPEG-8000 Monarch™ DNA Gel Extraction Kit M13KO7 helper phages 1-Step™ Ultra 3,3′,5,5′-Tetramethylbenzidine-enzyme-linked immunosorbent assay (TMB-ELISA) 6X DNA Gel Loading Dye Agar-Agar Carl Roth Agarose 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) Roti-Safe Gel Stain Tryptone/Peptone ® 20 TweenYeast extract ®-Prep Plasmid MINI RotiGeneJet PCR purification kit NotI and NcoI to both ends of the vNAR gene. RevRan_13-20 primers contain NNK repeats used to randomize the CDR3 region of the vNAR during PCR. The oligonucleotides used in this protocol are listed in Toptable Centrifuge 5427R Electrophoresis gel tank and power pack ® (Micropulser™) Electroporator Gene PulserFusion FX Gel Documentation New Brunswick Innova 4200 Incubator NanoDrop 1000 Spectrophotometer PCR Cycler Eppendorf Mastercycler SpectraMax M Series Multi-Mode Microplate Readers Vector NTI Advance 11GraphPad Prism version 7 Software CLC Main Workbench 8.0.1 pelB) signal sequence responsible for periplasmic transport of the fusion proteins. It also codes for an ampicillin resistance by β-lactamase for antibiotic selection. The antibody-pIII fusion protein can be overexpressed by Isopropyl β-D-1-thiogalactopyranoside (IPTG) induction through the lactose β-galactosidase (LacZ) promotor. Finally, the vector contains the bacterial ColE1 ori and the intergenic region of phage f1 as the origin of replication. The plasmid map and the used restriction enzyme NcoI and NotI locations are depicted in The phagemid pSEX81 is used for the construction of the library and preparation of phage antibody particles. This vector is designed for the expression of functional recombinant scFv-pIII fusion proteins on the surface of M13 bacteriophages. It further contains the bacterial pectate lysate , 5 ng of template DNA (HEL-5A7) and finally add 1 µL of each 10 µM primers . Use ForFR1_NcoI as forward primer and one of the randomized reverse primers (RevRan_12 to RevRan_23) for each reaction. Mix well and briefly centrifuge the contents.Perform PCR with the following set up: initial denaturation at 94 °C for 2 min. Then proceed for 20 cycles as follows: 5 s at 98 °C, annealing at 60 °C for 15 s and elongation of fragments for 15 s at 68 °C.Load PCR samples onto a 1% agarose gel and run the gel at 110 V. Inspect the gel under UV light. Excise the bands around 350 bp length and extract them from the agarose gel using the DNA gel extraction kit, according to manufacturer’s protocol. Elute the DNA of each PCR reaction with 10 µL nuclease-free water.Use the eluted PCR product for a second PCR to introduce restriction digestion sites. Proceed as it is described in step 2, use ForFR1_NcoI and RevFR4_NotI as primers for the reaction.NcoI and NotI. For this, prepare a total volume of 50 µL digestion reaction for the PCR products (at least 4 µg) by adding 2 µL (20 units) of each enzyme NcoI and NotI, 5 µL of 10X NEB3 buffer and adjust the volume reaction up to 50 µL with nuclease-free water. For the plasmid, digest 20 µg of the pSEX81 vector, add 15 µL of 10X NEB3 buffer, 10 µL (100 units) of each enzyme NcoI and NotI and add nuclease-free water up to 150 µL. Incubate for 3 h at 37 °C following 10 min at 65 °C.Digest both, the PCR products and the pSEX81 plasmid with After restriction digestion, purify DNA fragments using GeneJet PCR purification kit according to the manufacturer’s instruction. Elute each purified digested sample in 20 µL water.2O. Determine the concentration by Nanodrop 1000 spectrophotometer.PAUSE STEP: The extracted and purified digested DNA fragments can be stored at −20 °C until use.Run the digested plasmid on a 1% agarose gel and cut out the target band quickly with a scalpel on a UV-transilluminator table. Purify the DNA fragments from the agarose gel using the Monarch™ DNA Gel Extraction Kit, following the manufacturer’s instructions. Elute in 100 µL preheated sterile HLigate the digested fragments by preparing following material: Calculate the amount of needed insert for 100 ng of the plasmid with these molar ratios: 1:0.5, 1:1, 1:2 vector: insert. Add 1 µL of ligase, 1 µL of Ligase buffer (10X) and water to 10 µL. Keep at 16 °C overnight following 10 min at 65 °C.−2, 10−3, and 10−4 dilutions on 2X TY-GA agar plates and incubate overnight at 37 °C.Transform 2 µL of ligation reaction by electroporation into TG1 cells. Plate 100 µL of 10Measure the cloning efficiency by colony PCR with For_pSEX and Rev_pSEX primers. For this, pick 50 individual clones, mix each clone with 20 µL of water and cook them at 95 °C for 10 min. Later centrifuge them and take 2 µL of the supernatant as a template for the PCR.PAUSE STEP: After stopping the reaction, the mix can be stored at −20° C until further use.Based on the result of cloning efficiency, prepare the final ligation reaction for the best-determined ratio. For each insert with specific CDR3 length, add 2 µg of plasmid and an appropriate amount of each insert into 100 µL reaction volume. Incubate at 16 °C for 16 h, heat for 10 min at 65 °C and purify using GeneJet PCR purification kit. Elute each reaction in 20 µL and pool all the sample together.Time for Completion: 3 daysCRITICAL STEP: In order to increase the transformation efficiency, prepare electrocompetent cells on the same day of transformation and avoid freezing the cells before transformation.Time for Completion: two days or four days including preparation of competent cellsCRITICAL STEP: Chill all the material including HEPES, glycerol, H2O, pipettes and tips at 0 °C. Centrifuge and rotor must be precooled to 2 °C.12.E. coli TG1 from 2X TY-agar plate into 20 mL 2X TY medium in a 125 mL flask and incubate at 37 °C with vigorous shaking at 220 rounds per min (rpm) overnight.Pick a fresh clone of 13.600 at around 0.1) and incubate at 37 °C and 180 rpm until an optical density OD600 of 0.7.The next day, add the overnight culture to 1 L fresh 2X TY in a 5 L flask . Add a sterile magnetic stir bar to each bottle to completely dissolve the pellets on a magnetic stirrer.Remove the supernatant of both bottles carefully and re-suspend each pellet in 500 mL cold H17.2O/HEPES.Centrifuge for 10 min and repeat the previous step with another 500 mL cold H18.Discard the supernatant and resuspend each pellet in 50 mL cold glycerol/HEPES. Pool the bacteria suspension of both bottles into a fresh sterile centrifugation bottle without transferring the stir bars.19.Centrifuge for 15 min. Remove the supernatant and re-suspend the pellet in 1 mL cold glycerol/HEPES (see reagent setup).20.Distribute this volume into 300 µL aliquots. Use immediately for transformation.The protocol described here is based on the method from the Sidhu and co-workers ,24.CRITCRITICAL STEP: Cool at least 6 cuvettes and slides of the gene pulser down to 2 °C. For each control and test sample, prepare 0.950 mL of SOC in 2 mL tubes and 5 sterile 100 mL Erlenmeyer flasks with 25 mL SOC each for the samples.21.Apply this setting for the gene pulser: 2.5 kV and 25 µF.22.For negative control, dry the aluminum electrodes of the first cuvette with tissue paper, add 50 µL of electrocompetent cells to the cuvette, put the cuvette in the holder, push in into the gene pulser and start the electric pulse. Now, the cells are very fragile and you need to add pre-warmed SOC medium immediately. Rinse the cuvette a few times with SOC medium and put it quickly into the incubator.23.For the positive control add 1 µL of highly pure pSEX81 plasmid to 50 µL of the cells and perform the electroporation as described above.24.E. coli. Gently tab the bottom of cuvette on the bench to avoid bubble formation. After electroporation, immediately transfer the cells into the 125 mL flask and wash the cuvette with the prewarmed medium.Add 10–15 µL of purified ligation mixture to 300 µL of electrocompetent 25.Repeat step 24 for the other ligation reactions.26.Shake the entire samples at 37 °C in 220 rpm for 45–60 min.27.−2 to 10−5 dilutions of the library, 10−1 and 10−2 dilutions of the positive control and 100 µL of the undiluted negative control on 2X TY-GA plates.Pool the library and plate 100 µL of 1028.g for 10 min and discard the supernatant. Resuspend the pellet in 5 mL SOC medium and plate on five 150-mm 2X TY-GA plates.Centrifuge the rest of library suspension at 3220× 29.Incubate the plates overnight at 37 °C.30.−3 and 10−4 dilutions of the cells on 2X TY-GA agar plates.To estimate the transformation titer, plate 100 μL of 1031.The transformation titer is calculated as:32.g, 15 min), and resuspend the pellet in 1000 μL of 2X TY medium. Plate the suspended bacteria on a 150 mm diameter 2X TY-GA agar plate and incubate overnight at 37 °C.Centrifuge the remaining cells 35.600 should be approximately 0.1. Grow the culture at 37 °C, 250 rpm in a 2 L glass flask until OD600 = 0.5 (approximately 1.5–2 h).Thaw aliquot of frozen antibody library on ice. Add it to 500 mL 2X TY-GA medium. The initial OD36.12 M13KO7 hyperphages to the culture and incubate at 37 °C without shaking for 15 min following 30 min vigorous shaking.Add 1 × 1037.g for 15 min and discard the supernatant.Spin the culture at 3200× 38.Resuspend pellets in 500 mL of 2X TY-AK medium.39.Grow for 16–20 h at 28 °C, 250 rpm in a 2 L glass flask.40.g and 4 °C.The next day, centrifuge the culture for 20 min at 5500× 41.Add 1/4 volume of PEG/NaCl solution (see Reagent setup) to precipitate the produced phage binder from the culture supernatant.42.g and 4 °C.Following four hours of incubation on ice, pellet the antibody phages by centrifugation for 60 min at 5500× 43.Re-suspend the white phage pellet in 1 mL PBS.44.E. coli particles and bacterial debris, centrifuge the solution three times for 5 min.To remove 45.Collect the supernatant and transfer it to a new 1.5 mL tube containing 250 µL of PEG/NaCl.46.g for 10 min and discard the supernatant.Incubate it on ice for 30 min. Then pellet the precipitated phages by centrifugation at 17,900× 47.g and carefully remove the remaining supernatant.To remove the final traces of PEG, centrifuge again for another 2 min at 17,900× 48.Re-suspend the phages in 0.5 mL PBS. This supernatant contains the phage particles. Store them at 4 °C. These phages are used for the first round of panning.49.For the first panning round, use 4 µg protein/well per panning, for the following rounds use 1 µg protein/well for more stringent conditions. Dissolve the antigen and incubate in a microtiter plate well overnight at 4 °C. Coating conditions (incubation temperature and coating buffer) should be set-up for the antigen of interest. Prepare overnight culture of TG1 cells grown on minimal medium (M9) by transferring 1 colony from fresh culture to 5 mL LB. Growth of TG1 on minimal medium selects for cells with F’ factor.50.3 (pH 9.6) on a 96-well well plate incubated at 4 °C overnightFor biopanning of the synthetic library, coat 2 µg of the antigen TNF alpha in 150 µL of 50 mM NaHCO51.The following day, remove the coating solution from the plate by tapping on a clean tissue. Wash the coated well three times with PBST in an ELISA washer or squirt bottle and block with 300 µL MPBST for 2 h at room temperature (RT).52.After blocking, wash the well once with PBST and one to three times with PBS.53.11 antibody phages in 200 µL blocking buffer for 1 h without shaking and 1 h at RT and gentle agitation (200 rpm). Meanwhile, inoculate 5 mL 2X TY with 500 µL of the overnight culture of E. coli TG1 and grow at 37 °C, 250 rpm to an OD600 0.5. The culture can be kept at 4 °C until use. For the following round of panning change the blocking buffer to BSA-PBST to eliminate nonspecific binding of phages to the blocking buffer.For binding of the antibody phages, incubate 1054.® 20 concentration by 0.1% for each washing step. For the subsequent panning rounds, increase the washing steps to 15 (round 2) and 20 (round 3 and 4) steps with PBST, respectively.Discard the unbound phages by washing 10 times with PBST and 10 times with PBS for 2 min each. Increase the Tween55.Elute phage particles by using 50 µL trypsin solution . Combine both suspensions.Add 100 µL of a log-phased 57.−2 to 10−4 in 2X TY medium) on 2X TY-GA agar plates and incubated overnight at 37 °C.After infection, plate 100 µL of the total volume from step 56 in serial dilutions  and mix thoroughly.62.Prepare 100 µL aliquots and store at –20 °C until further use.63.For the subsequent round of panning, the eluted phages should be packaged and reamplified. Inoculate 1 mL of 2X TY-GA medium with 20 µL of the glycerol stock obtained after panning. Grow the culture for about 3 h at 37 °C 200 rpm.64.9 cfu (colony forming units) of M13KO7 helper phages and incubate for 1 h at 37 °C and 200 rpm.Infect the cells with 2 × 1065.g and 4 °C. Discard the supernatant.To remove the medium, centrifuge the infected culture for 30 min at 3200× 66.Suspend the pellet in 500 µL 2X TY/AK medium and incubate for 16 h at 30 °C 200 rpm for phage production.67.The next day, pellet the bacteria twice by centrifugation for 10 min at 3200 rpm and 4 °C to precipitate the phages. Add 1/5 volume of PEG/NaCl solution to the supernatant and incubate for 1 h on ice.68.g and 4 °C.To pellet the phages, centrifuge the solution for 1 h at 3200× 69.Resuspend the white phage pellet in 100 µL PBS and store at 4 °C.70.Make serial dilutions of the phage suspension in PBS. The number of eluted phage depends on several parameters .71.−6, 10−7, 10−8) and incubate 30 min at 37 °C.Infect 50 µL bacteria with 10 µL of different phage dilutions : 3–4 days .75.3 (pH 9.6) in duplicate for each round of panning and incubated overnight at 4 °C.For this, coat a 96-well plate with 50 ng of TNF alpha in 100 µL 50 mM NaHCO76.The following day, wash the coated wells three times with 200 µL PBST and then block with 300 µL MPBST for 2 h at RT.77.9 cfu phage amplificate from each panning round to each well and incubate the plate for 1.5 h at RT.After three washing steps with each 200 µL PBST, add 1078.After incubation, wash the wells three times with 200 µL PBST79.Add 100 µL of a 1:100 dilution of anti-pVIII monoclonal antibody in blocking buffer and incubate for an additional 1 h at RT.80.Wash the plates as above, and add 100 µL of a 1:10,000 dilution of the secondary antibody anti-mouse IgG peroxidase conjugate in blocking buffer and incubate for a further hour at RT.81.After three washing steps add 100 µL of 1-Step™ Ultra TMB-ELISA substrate for color development to each well.82.2SO4).Stop the reaction by the addition of 100 µL of 1 M sulfuric acid . Seal the plate with a breathable sealing film.89.Incubate overnight at 37 °C and 250 rpm.90.600 of 0.7–0.9. To the rest of the overnight culture add 10 µL glycerol, mix well, and store at −80 °C.Transfer 10 µL of overnight culture to a new 96 well plate and add 150 µL 2X TY-GA. Incubate at 37 °C and 250 rpm for approximately 2–3 h to reach an OD91.Centrifuge the plate for 10 min at 4000 rpm, remove the medium carefully and add 150 µL 2X TY medium, suspend the pellet and centrifuge again.92.Add 150 µL 2X TY-A with 100 µM IPTG and incubate overnight at 30 °C and 250 rpm.93.Centrifuge for 10 min at 4000 rpm. Transfer the antibody fragment containing supernatant to a new polypropylene plate (first supernatant) and store at 4 °C.94.Add 100 µL of the periplasmic buffer to the pellet, resuspend and incubate on ice for 30 min.Centrifuge the plate, remove the supernatant (second supernatant) and combine it with the first supernatant. Keep at 4 °C until the next step.The soluble vNARs are produced in fusion with pIII of the phagemid  or PBS for coating. For the positive control, coat 100 µL of 1 mg/mL of lysozyme and 50 ng of BSA as a negative control. Incubate overnight at 4 °C or 1 h at 37 °C.Coat 50 ng of antigen into 96 well plates in 100 µL of carbonate buffer  with 100 µL of blocking buffer and add them to coated wells. Incubate for 90 min at RT.98.Wash 3 times with PBST.99.As a primary antibody, use a 1:1000 dilution of an anti pIII monoclonal antibody and incubate for 1 h at RT.100.Add secondary antibody and proceed with the steps 80–83 of 101.To determine the nucleotide sequence of plasmid DNA, DNA samples should be sent to sequencing. The sequencing of our sample was performed at GATC BIOTECH AG . The resulting data of the sequencing can be analyzed using the software of Vector NTI or CLC Main Workbench 8 or any similar software.After the amplification of DNA fragment using the reverse randomized primer with different lengths, you will get different bands from 350 to 400 bp based on each reverse primer used for the PCR reaction see . The PCRColony PCR is used to evaluate the quality of cloning and transformation and to determine whether the insert is present in the plasmid. The position of primers on the plasmid and expected size of the fragments are shown in DpnI in case the parental DNA plasmid was propagated in standard E. coli strains. Also, frameshift mutations can be detected in amplified fragments, although the primers used contain only multiples of 3 codons. These errors introduced by oligonucleotides can lead to nonsense codon (clone 2) or very different sequence in CDR3 and the rest of vNAR framework 4 (clone 3). Such framework shifts occurred in 40% of the sequenced clones. Errors in primers also can lead to deletion of conserved cysteine, which prevents the formation of the stabilizing disulphide bond (clone 4). Presence of amber stop codon (clone 5) occurred in 8% of the sequenced clones. As long as SupE strains of E. coli are used in phage display, this codon can be mostly read as glutamine (Q). Finally, other clones will represent the array used in primers with different length and two conserved cysteines (clone 6 to clone 13). By using this protocol and by ensuring that the library is large enough, the number of clones with the above-mentioned problems is rather low, and a sufficient amount of binding protein against the antigen of interest can be obtained.To further evaluate the quality of the library, the distribution of amino acids in the artificial CDR3 libraries needs to be assessed via sequencing. As shown in ® 20 is also increased in subsequent panning round. In addition, non-specific elution may cause a problem. Rather harsh conditions are needed to elute strong binders, but they shall not harm the quality of the phages. The titer of the eluted phages is determined to evaluate the enrichment and thereby the quality of this panning step after each round of panning. After using standard conditions for elution , a polyclonal ELISA was performed on the plate used for biopanning. Surprisingly, a high ELISA signal (data not shown) corresponding to a high number of phages that are still bound either specifically to the antigen or un-specifically to the well. Therefore, we tested several different methods to elute the phages after biopanning : Dissolve 400 g of glucose in 1 L of deionized water. Filter through0.2 µm filter. Can be stored at 4 °C for several months.2X TY medium pH 7.0: 1.6% (w/v) tryptone, 1% (w/v) yeast extract, 0.5% (w/v) NaCl)2X TY-GA: 2X TY, 100 mg/mL ampicillin, 100 mM glucose2X TY-GA agar plates: Dissolve 10 g tryptone, 5 g yeast extract, and 10 g NaCl in 1L water. Add 15 g agar-agar and autoclave. Cool down below 50 °C, add 1 mL of filter-sterilized ampicillin (100 mg/mL) and 50 mL of 40% (w/v) filter sterilized glucose. Mix with gentle stirring and fill one-third of 100 mm or 150 mm diameter polystyrene Petri dishes. Keep the plates at 4 °C.SB medium pH 7.0: Dissolve 30 g tryptone, 20 g yeast extract and 10 g MOPS (3-[N-morpholino]–propanesulfic acid) in 1L water. Dissolve and autoclave.4 (autoclaved), 0.1 mL of 1 M CaSO4 (autoclaved), 5 mL of 40% glucose, and 0.25 mL of 1% thiamine HCl (filter-sterilized). Mix with gentle stirring and pour on polystyrene Petri dishes. Keep the plates at 4 °C.Minimal medium agar plate: Add 5.6 g 5X M9 salt and 15 g agar to 500 mL of deionized water and autoclave. When cooled to 50-45° C, add 1 mL of 1 M MgSO2.SOC medium: Add 20 g of tryptone, 5 g of yeast extract and 0.5 g of NaCl to 950 mL of deionized water. Shake well and add 10mL of a 250 mM KCl. Adjust the pH of the medium to 7.0. autoclave for 20 min. Add 5 mL of a sterile solution of 2 M MgClAgarose electrophoresis gel: For 1% agarose gel, use 1 g of agarose and 100 mL TAE buffer . Change the amount of agarose as needed to make 1.5 or 2% gels.2O/HEPES: Add 1 mL of sterile HEPES 1M to 1 L of autoclaved ultra-pure water.HGlycerol/HEPES: Add 1 mL of sterile HEPES 1M to 1L 10% glycerol.2. Dissolve 1.5 g of TrisBase, 8 g NaCl and 0.15 g CaCl2 in 1 L of deionized water. Adjust to pH 7.4 and autoclave.TBSC buffer: 10 mM Tris pH 7.4, 137 mM NaCl, 1 mM CaClPEG solution: 20% PEG, 2.5 M NaCl. Dissolve 100 g of PEG 8000 and 73 g of NaCl in 500 mL of deionized water. Filter through 0.2 µm filter.Trypsin solution: Dissolve trypsin powder at 10 mg/mL in TBSC (trypsin stock). Freeze in 20 µL aliquots in liquid nitrogen. This can be stored at −20 °C for several months. For the experiment, dissolve 100 µlL of trypsin stock in 10 mL of TBSC (trypsin solution).® 20 up to 1X PBS + 1% (v/v) Tween® 20.PBST: 1X PBS + 0.1% (v/v) TweenMPBST: 2% skimmed milk in PBST, prepare freshly.Panning block solution: MPBST or BSA-PBST: 1% (w/v) BSA in PBST, prepare freshly.Periplasmic buffer: 100 mM Tris; 0.5 M Sucrose; 1 mM EDTA; pH 8.0