STARR-seq and UMI-STARR-seq: Assessing Enhancer Activities for Genome-Wide-, High-, and Low-Complexity Candidate Libraries.
The identification of transcriptional enhancers and the quantitative assessment of enhancer activities is essential to understanding how regulatory information for gene expression is encoded in animal and human genomes. Further, it is key to understanding how sequence variants affect enhancer function. STARR-seq enables the direct and quantitative assessment of enhancer activity for millions of candidate sequences of arbitrary length and origin in parallel, allowing the screening of entire genomes and the establishment of genome-wide enhancer activity maps. In STARR-seq, the candidate sequences are cloned downstream of the core promoter into a reporter gene's transcription unit (i.e., the 3' UTR). Candidates that function as active enhancers lead to the transcription of reporter mRNAs that harbor the candidates' sequences. This direct coupling of enhancer sequence and enhancer activity in cis enables the straightforward and efficient cloning of complex candidate libraries and the assessment of enhancer activities of millions of candidates in parallel by quantifying the reporter mRNAs by deep sequencing. This article describes how to create focused and genome-wide human STARR-seq libraries and how to perform STARR-seq screens in mammalian cells, and also describes a novel STARR-seq variant (UMI-STARR-seq) that allows the accurate counting of reporter mRNAs for STARR-seq libraries of low complexity. © 2019 The Authors. Basic Protocol 1: STARR-seq plasmid library cloning Basic Protocol 2: Mammalian STARR-seq screening protocol Alternate Protocol: UMI-STARR-seq screening protocol-unique molecular identifier integration Support Protocol: Transfection of human cells using the MaxCyte STX scalable transfection system.
['Neumayr|Christoph|C|', 'Pagani|Michaela|M|', 'Stark|Alexander|A|', 'Arnold|Cosmas D|CD|']
D000818:Animals D003001:Cloning, Molecular D004742:Enhancer Elements, Genetic D005821:Genetic Techniques D015698:Genomic Library D006367:HeLa Cells D006801:Humans D010957:Plasmids D014162:Transfection
STARR-seq UMI-STARR-seq enhancer gene expression MPRA cis-regulatory element functional genomics gene regulation massively parallel reporter assay transcriptional regulation