3'-Seq

3'-Seq

3'-Seq was designed to measure the abundance of 3'-UTR isoforms quantitatively in a wide array of human tissue types. The first cDNA strand is generated by reverse-transcribing total RNA using an oligo(dT) primer containing a VN-anchor (where V is dA, dC, or dG), P5 adapter sequence, a uridine, and biotin that is bound to a streptavidin-coated magnetic bead. The second cDNA strand is synthesized using DNA polymerase I. To initiate nick translation with DNA polymerase I, RNase HII is used to introduce a nick at the uridine. This creates another nick that is 50–75 bases away from the 3' end. A blunt end is created at the position of the new nick, and double-stranded P7 adapters are ligated at this position. The double-stranded cDNA is PCR-amplified, purified, and ready for sequencing.

Pros:

• Measures 3' UTR isoform abundance to detect protein expression differences in multiple tissue types

Cons:

• Technically challenging; nick translation requires precise conditions

• 3'-Seq: Lianoglou S., Garg V., Yang J. L., Leslie C. S. and Mayr C. (2013) Ubiquitously transcribed genes use alternative polyadenylation to achieve tissue-specific expression. Genes Dev 27: 2380-2396

1. Bangru S. and Kalsotra A. Advances in analyzing RNA diversity in eukaryotic transcriptomes: peering through the Omics lens. F1000Research. 2016;5:2668
2. Miura P., Sanfilippo P., Shenker S. and Lai E. C. Alternative polyadenylation in the nervous system: to what lengths will 3' UTR extensions take us? Bioessays. 2014;36:766-777

1. Kralovicova J., Knut M., Cross N. C. and Vorechovsky I. (2015) Identification of U2AF(35)-dependent exons by RNA-Seq reveals a link between 3' splice-site organization and activity of U2AF-related proteins. Nucleic Acids Res