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Methods. 2017 Aug 15;126:86-94. doi: 10.1016/j.ymeth.2017.06.003. Epub 2017 Jun 8.

Genome-wide profiling of the 3' ends of polyadenylated RNAs.

Author information

1
Department of Developmental Biology, Sloan-Kettering Institute, New York, NY 10065, USA; Louis V. Gerstner, Jr. Graduate School of Biomedical Sciences, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA.
2
Department of Biology, University of Nevada, Reno, Reno, NV 89557, USA.
3
Department of Developmental Biology, Sloan-Kettering Institute, New York, NY 10065, USA; Louis V. Gerstner, Jr. Graduate School of Biomedical Sciences, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA. Electronic address: laie@mskcc.org.

Abstract

Alternative polyadenylation (APA) diversifies the 3' termini of a majority of mRNAs in most eukaryotes, and is consequently inferred to have substantial consequences for the utilization of post-transcriptional regulatory mechanisms. Since conventional RNA-sequencing methods do not accurately define mRNA termini, a number of protocols have been developed that permit sequencing of the 3' ends of polyadenylated transcripts (3'-seq). We present here our experimental protocol to generate 3'-seq libraries using a dT-priming approach, including extensive details on considerations that will enable successful library cloning. We pair this with a set of computational tools that allow the user to process the raw sequence data into a filtered set of clusters that represent high-confidence functional polyadenylation sites. The data are single-nucleotide resolution and quantitative, and can be used for downstream analyses of APA.

KEYWORDS:

3′ UTR; 3′ end; APA; Genome-wide profiling; Polyadenylation

PMID:
28602807
PMCID:
PMC5583017
DOI:
10.1016/j.ymeth.2017.06.003
[Indexed for MEDLINE]
Free PMC Article

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