Simple and efficient profiling of transcription initiation and transcript levels with STRIPE-seq

Abstract

Accurate mapping of transcription start sites (TSSs) is key for understanding transcriptional regulation. However, current protocols for genome-wide TSS profiling are laborious and/or expensive. We present Survey of TRanscription Initiation at Promoter Elements with high-throughput sequencing (STRIPE-seq), a simple, rapid, and cost-effective protocol for sequencing capped RNA 5′ ends from as little as 50 ng total RNA. Including depletion of uncapped RNA and reaction cleanups, a STRIPE-seq library can be constructed in about 5 h. We show application of STRIPE-seq to TSS profiling in yeast and human cells and show that it can also be effectively used for quantification of transcript levels and analysis of differential gene expression. In conjunction with our ready-to-use computational workflows, STRIPE-seq is a straightforward, efficient means by which to probe the landscape of transcriptional initiation.

Global approaches for profiling transcription initiation

“…TSRT was later adapted to mapping of TSSs at single-cell resolution in methods such as C1-CAGE ( Kouno et al, 2019 ), Tn5Prime ( Cole et al, 2018 ), and low-input Parallel Analysis of RNA Ends (nanoPARE) ( Schon et al, 2018 ). Finally, Survey of TRanscription Initiation and Promoter Elements with high-throughput sequencing (STRIPE-seq) ( Policastro et al, 2020 ) removed the need for semi-suppressive PCR or tagmentation for modest RNA input amounts, and further reduced the prevalence of artifactual reads through improved oligo design and methodological optimizations.…”

“…We therefore suggest selecting a threshold within the inflection point of the promoter proximal fraction curve to balance removal of likely artifacts with retention of weak true TSSs. We found that, in STRIPE-seq data from yeast and human cells, a threshold of three raw counts per TSS is a suitable threshold for TSS retention ( Policastro et al, 2020 ).…”

Flexible analysis of TSS mapping data and detection of TSS shifts with TSRexploreR

“…We recently introduced a new TSS mapping method termed Survey of TRanscription Initiation at Promoter Elements with high-throughput sequencing (STRIPE-seq) (14), a rapid, efficient, simple, and cost-effective TSS profiling approach compatible with limited input amounts. In parallel, we developed software to streamline analysis of STRIPE-seq data, as well as data resulting from other popular methods.…”

Systematic comparison of experimental assays and analytical pipelines for identification of active enhancers genome-wide

“…1a). To enrich for RNA populations of interests, these assays implement various experimental strategies, including nuclei/chromatin isolation [8][9][10][11]18 , nuclear run-on [8][9][10] or metabolic labeling 19,20 with biotin or bromo-tagged nucleotides and affinity purification, Pol II immunoprecipitation 18 , size selection 15,16 , and enzymatic elimination of non-capped RNAs [8][9][10]16 , or chemical tagging of capped RNAs [12][13][14] . We summarized key experimental steps of both 5′ and 3′ assays in Fig.…”

“…1a). We named the 8 assays (GRO 9 /PRO-cap 10 , CoPRO 8 , Start-seq 11 , CAGE 12 , RAMPAGE 13 , NET-CAGE 14 , csRNA-seq 15 , and STRIPE-seq 16 ) from the former category as 5’ assays, because these assays enrich for active 5’ TSSs of promoters and enhancers (Fig. 1a).…”