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.

Keywords: MPRA; STARR-seq; UMI-STARR-seq; cis-regulatory element; enhancer; functional genomics; gene expression; gene regulation; massively parallel reporter assay; transcriptional regulation.

Figure 1

Step‐wise schematic overview of STARR‐seq and UMI‐STARR‐seq. A highly complex library insert (Illumina adapter flanked candidate fragments) is cloned into the 3′UTR of the reporter transcription unit of the STARR‐seq screening plasmid (Basic Protocol 1). After STARR‐seq library transfection into cells, active enhancers transcribe themselves as part of the 3’UTR of the reporter transcripts. The reporter transcripts are isolated as part of the cellular mRNA (isolation of mRNA) and selectively reverse transcribed using a reporter‐specific RT primer (green arrow). Standard STARR‐seq (Basic Protocol 2; left): To selectively amplify reporter cDNAs and not residual input DNA (library plasmid), a nested 2‐step PCR strategy is employed. During the junction PCR step, reporter cDNAs are specifically amplified using a forward primer (junction primer) that exclusively binds to the spliced reporter cDNA. Using the Illumina index primers (i5 and i7), the candidate sequences (Illumina adapter flanked) are amplified during the sequencing‐ready PCR step and subjected to deep sequencing. UMI‐STARR‐seq (Alternate Protocol; right): For low‐complexity candidate libraries, unique molecular identifiers (UMI) can be introduced prior to amplification, allowing the precise counting of reporter mRNAs (Alternate Protocol). To render the reporter cDNAs competent for UMI introduction, second‐strand synthesis is performed with a reporter‐specific forward primer, resulting in double‐stranded reporter DNA. The UMI is introduced by linear PCR with a modified Illumina i7 primer that harbors the UMI at the position of the i7 index. Up to this step, no amplification occurred, such that the UMIs identify individual reporter mRNAs. Only now, the UMI bearing reporter DNAs are amplified, first by the junction PCR and then by the sequencing‐ready PCR. Note that in UMI‐STARR‐seq, indexing is only possible using the i5 index, as the i7 index is replaced by the UMI. Consequently, the UMI is read as Index 1 during deep sequencing on an Illumina platform. STARR‐seq signal over input directly and quantitatively reports on the enhancer activity. ORI, bacterial origin of replication; ORF, open reading frame included in the reporter transcript with no specific function except RNA stabilization. pA site, poly‐adenylation site; P5 and P7, P5 (forward) and P7 (reverse) Illumina adapters.

Figure 2

Test PCR on 1% agarose gel (lanes): left lane, Fermentas GeneRuler 1 kb plus; (1) 10 µl of sequencing‐ready PCR with five cycles; (2) 10 µl of sequencing‐ready PCR with nine cycles; and (3) 10 µl of sequencing‐ready PCR with nine cycles of minus RT.

Figure 3

Sequencing‐ready PCR on 1% agarose gel (lanes): left lane, Fermentas GeneRuler 1 kb plus; (1) 10 µl of the pooled PCRs; (2) 10 µl of the supernatant, taken after PCR has bound to the beads; and (3) 250 ng cleaned‐up PCR product.