STARR‐seq and UMI‐STARR‐seq: Assessing Enhancer Activities for Genome‐Wide‐, High‐, and Low‐Complexity Candidate Libraries

Abstract

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

Assessing genome-wide dynamic changes in enhancer activity during early mESC differentiation by FAIRE-STARR-seq

“…1A). A similar approach has been described, however in that case UMIs are introduced in a first PCR cycle after the reverse transcription step (Neumayr et al ., 2019). The introduction of UMIs allows one to distinguish between biological replicates and PCR duplicates that can dramatically distort the relative quantities of individual fragments within a library (Islam et al ., 2014).…”

“…When we started our project, UMIs were not part of the STARR-seq protocol. However, in the meantime a similar approach has been proposed for low complexity libraries where UMIs are introduced in the first PCR cycle (Neumayr et al ., 2019). By applying UMIs, we could not only identify active enhancers, but also show that specific sequence features are associated with activity levels (Fig.…”

Parallel functional testing identifies enhancers active in early postnatal mouse brain

“…We observed some variability in amplicon RNA dropout across replicates, likely due to a combination of transduction efficiency variability across animals, sensitivity recapturing amplicons with low viral representation, and PCR stochasticity 52 . While not an obvious issue here, effect of PCR-based clonal amplification bias in other MPRAs has been shown to be reduced with the addition of barcodes and unique molecular identifiers to the reporter construct 53 . Amplicons in this assay were approximately ~900 bp long, representing some of the longer sequences tested in MPRAs to date.…”

“…No reuse allowed without permission. molecular identifiers to the reporter construct 53 . Amplicons in this assay were approximately ~900 bp long, representing some of the longer sequences tested in MPRAs to date.…”

“…As additional quality control, reads were also aligned to the mouse genome (GRCm38) to check for cross-contamination of mouse DNA from the amplicon PCR. Aligned SAM files were processed using Picard tools (v.2.23.3) 53 : aligned reads were sorted by coordinates and converted to BAM files using SortSam , optical duplicates were flagged by MarkDuplicates , and BAM file indexing was conducted using BuildBamIndex . The number of de-duplicated reads within in silico PCR amplicon coordinates was counted using samtools view -F 0×400 -c (v1.7) 62 and reported as the raw amplicon count.…”