3' end sequencing protocols

Learn more about the techniques used for 3' end sequencing.

2P-Seq

In the 2P-Seq protocol, reverse transcription is accomplished by an anchored oligo(dT) primer. The products of reverse transcription and PCR amplification are expected to have 20 As preceding the 3' adapter. Libraries are sequenced in anti-sense direction with a custom primer requiring to reverse complement reads to pinpoint the 3' end.

Publications & data sets

Spies, N., Burge, C.B., Bartel, D.P. 3' UTR-isoform choice has limited influence on the stability and translational efficiency of most mRNAs in mouse fibroblasts. Genome Res 23, 2078-90 (2013). Link out

3'-Seq (Mayr)

In the 3'-Seq protocol by Mayr and colleagues, reverse transcription is accomplished by an anchored oligo(dT) primer. The products of reverse transcription and PCR amplification are expected to have 17 As preceding the 3' adapter. Libraries are sequenced in sense direction requiring removal of the 3' adapter sequence and preceding As to pinpoint the 3' end.

Publications & data sets

Singh, I. et al. Widespread intronic polyadenylation diversifies immune cell transcriptomes. Nat Commun 9, (2018) Link out

Lee, S.-H. et al. Widespread intronic polyadenylation inactivates tumor suppressor genes in leukemia. Nature 561, 127–131 (2018) Link out

Lianoglou, S., et al. Ubiquitously transcribed genes use alternative polyadenylation to achieve tissue-specific expression. Genes Dev 27, 2380-96 (2013). Link out

3'READS

3' region extraction and deep sequencing (3'READS) is a protocol that utilizes a special oligo(dT) primer ( 45 thymidines followed by 5 uridines) to capture poly(A) containing RNA fragments. After partial digestion of the poly(A) tail, the fragments are subjected to adapter ligation, reverse transcription, and PCR amplification before they are sequenced in anti-sense direction. The cleavage site is inferred as the first non-A of the 3' end of the read's reverse complement.

Publications & data sets

Yang, Y. et al. PAF Complex Plays Novel Subunit-Specific Roles in Alternative Cleavage and Polyadenylation. PLoS Genet 12, (2016) Link out

Publicly available data without associated publication.

Li, W. et al. Systematic Profiling of poly(A)+ Transcripts Modulated by Core 3’ End Processing and Splicing Factors Reveals Regulatory Rules of Alternative Cleavage and Polyadenylation. PLoS Genetics 11 (4), e1005166 (2015). Link out

Zheng, D. et al. Cellular stress alters 3′UTR landscape through alternative polyadenylation and isoform-specific degradation. Nat Commun 9, (2018) Link out

Li, W. et al. Alternative cleavage and polyadenylation in spermatogenesis connects chromatin regulation with post-transcriptional control. BMC Biol 14, (2016) Link out

Wang, R. et al. A compendium of conserved cleavage and polyadenylation events in mammalian genes. Genome Res 10, 1427-1441. (2018) Link out

3P-Seq

In the 3P-Seq protocol, a biotinylated adapter is ligated to the end of the poly(A) tail via splint-ligation and the poly(A) region is reverse transcribed with only dTTP. After removal of most of the poly(A) tail, adapters are ligated prior to reverse transcription and PCR amplification. Finally, the libraries are sequenced in anti-sense direction and the resulting reads are reverse complemented in order to pinpoint the 3' ends.

Publications & data sets

Nam, J. W. et al. Global analyses of the effect of different cellular contexts on microRNA targeting. Mol Cell 53, 1031-43 (2014). Link out

Jan, C. H., Friedman, R. C., Ruby, J. G. & Bartel, D. P. Formation, regulation and evolution of Caenorhabditis elegans 3’UTRs. Nature 469, 97–101 (2011). Link out

A-seq

In the A-seq protocol, reverse transcription is accomplished by an anchored oligo(dT) primer. The products of reverse transcription and PCR amplification are expected to have six As preceding the 3' adapter. Libraries are sequenced in sense direction requiring removal of the 3' adapter sequence and preceding As to pinpoint the 3' end.

Publications & data sets

Martin, G., Gruber, A. R., Keller, W. & Zavolan, M. Genome-wide analysis of pre-mRNA 3’ end processing reveals a decisive role of human cleavage factor I in the regulation of 3' UTR length. Cell Rep 1, 753–763 (2012). Link out

Gruber, AR. et al. Global 3’ UTR Shortening Has a Limited Effect on Protein Abundance in Proliferating T Cells. Nat Commun 5, 5465 (2014). Link out

Gruber, A. R., Martin, G., Keller, W. & Zavolan, M. Cleavage factor Im is a key regulator of 3’ UTR length. RNA Biol 9, 1405–1412 (2012). Link out

DRS

In the direct RNA sequencing (DRS) protocol, 3' ends of transcripts are hybridized to poly(dT)-coated flow cell surfaces where antisense strand synthesis is initiated. This has the advantage that no prior reverse transcription or cDNA amplification is needed.

Publications & data sets

Rehfeld, A. et al. Alternative polyadenylation of tumor suppressor genes in small intestinal neuroendocrine tumors. Front Endocrinol (Lausanne) 5, 46 (2014). Link out

Yao, C. et al. Transcriptome-wide analyses of CstF64-RNA interactions in global regulation of mRNA alternative polyadenylation. Proc Natl Acad Sci U S A 109, 18773–18778 (2012). Link out

Ji, X. et al. αCP Poly(C) Binding Proteins Act as Global Regulators of Alternative Polyadenylation. Mol Cell Biol 33, 2560–2573 (2013). Link out

Lackford, B. et al. Fip1 regulates mRNA alternative polyadenylation to promote stem cell self-renewal. EMBO J (2014). Link out

PAPERCLIP

To capture RNA 3'ends PAPERCLIP takes advantage of the specific interaction of poly(A)-binding protein (PABP) with the RNA's poly(A) tail. After crosslinking RNA-binding proteins to RNAs, the RNAs are fragmented, and RNA fragments crosslinked to PABP are purified with a specific antibody. The protein is then degraded and the RNA fragments are subjected to reverse transcription with a primer that includes sequencing adaptors. Subsequently the cDNA is circularized, the insert is amplified by PCR using primers specific for the adaptor sequences, and finally sequenced in sense direction.

Publications & data sets

Jereb, S. et al. Differential 3’ Processing of Specific Transcripts Expands Regulatory and Protein Diversity Across Neuronal Cell Types. Elife 7, (2018) Link out

Hwang, H.-W. et al. cTag-PAPERCLIP Reveals Alternative Polyadenylation Promotes Cell-Type Specific Protein Diversity and Shifts Araf Isoforms with Microglia Activation. Neuron 95, 1334-1349.e5 (2017) Link out

Hwang, H.W. et al. PAPERCLIP Identifies MicroRNA Targets and a Role of CstF64/64tau in Promoting Non-canonical poly(A) Site Usage. Cell Rep 15, 423-35 (2016) Link out

PAS-Seq

In the PAS-Seq protocol, reverse transcription is accomplished by an anchored oligo(dT) primer. The products of reverse transcription and PCR amplification are expected to have 20 As preceding the 3' adapter. Libraries are sequenced in anti-sense direction with a custom primer requiring to reverse complement reads to pinpoint the 3' end.

Publications & data sets

Shepard, P. J. et al. Complex and dynamic landscape of RNA polyadenylation revealed by PAS-Seq. RNA 17, 761–772 (2011). Link out

PAT-seq

For PAT-seq, adenylated RNA is extended by dNTPs using Klenow polymerase and a biotinylated anchor-oligo. Internal priming is avoided by a requirement for this 3' extension in subsequent steps. Tagged RNA is fragmented with RNase T1, the 3' end fragments are collected with streptavidin beads and ligated to a 5' linker. After reverse transcription fragments are selected by size to ensure that sequencing reaches the poly(A) tail. Subsequently they are PCR amplified and sequenced in sense direction, requiring removal of 3' As to pinpoint the 3' ends.

Publications & data sets

Elewa, A. et al. POS-1 Promotes Endo-mesoderm Development by Inhibiting the Cytoplasmic Polyadenylation of neg-1 mRNA. Dev Cell 34, 108–118 (2015). Link out

PolyA-seq

Library preparation for the PolyA-seq protocol requires (1) Reverse transcription, primed with an oligo(dT) sequence succeeding an universal sequence used as PCR anchor, (2) second strand synthesis with random hexamers linked to a second PCR primer, and (3) PCR amplification. Sequencing is accomplished in anti-sense orientation with a primer ending in 10 Ts; the resulting reads need to be reverse complemented in order to pinpoint the mRNA cleavage site.

Publications & data sets

Derti, A. et al. A quantitative atlas of polyadenylation in five mammals. Genome Res 22, 1173–1183 (2012). Link out

Batra, R. et al. Loss of MBNL Leads to Disruption of Developmentally Regulated Alternative Polyadenylation in RNA-Mediated Disease. Mol Cell 56, 311–322 (2014) Link out

QuantSeq_REV

QuantSeq is the method implemented by Lexogen, starting from total RNA and requiring a turnaround time of a few hours. The reverse transcription of polyadenylated RNAs is done with an oligo(dT) primer that contains the 3’ adaptor. Second strand synthesis is then carried out by random priming, with oligos that also contain the 5’ adaptor. The library is amplified and sequenced, in the case of QuantSeq REV starting from the last nucleotide of the mRNA in antisense direction.

Publications & data sets

Publicly available data without associated publication.

SAPAS

In the SAPAS protocol, reverse transcription is accomplished by an anchored oligo(dT) primer. The products of reverse transcription and PCR amplification are expected to have the sequence AAAAAAGAAAAAAGAAAAAA preceding the 3' adapter. Libraries are sequenced in anti-sense direction with a regular primer requiring to trimm 20 nucleotides from the 5' end of reads and to reverse complement reads to pinpoint the 3' end.

Publications & data sets

You, L. et al. APASdb: A Database Describing Alternative poly(A) Sites and Selection of Heterogeneous Cleavage Sites Downstream of poly(A) Signals. Nucl Acids Res 43 (Database issue), D59–67 (2015). Link out

Fu, Y. et al. Differential genome-wide profiling of tandem 3’ UTRs among human breast cancer and normal cells by high-throughput sequencing. Genome Res 21, 741–747 (2011). Link out