3′-Seq
Quantitatively Measure Abundance of 3’UTR Isoforms
3′-Seq was designed to measure the abundance of 3′-UTR isoforms quantitatively in a wide array of human tissue types (Lianoglou et al., 2013). 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.
Advantages:
- Measures 3’UTR isoform abundance to detect protein expression differences in multiple tissue types
Disadvantages:
- Technically challenging; nick translation requires precise conditions
Reagents:
Illumina Library prep and Array Kit Selector
Reviews:
Bangru S. and Kalsotra A. Advances in analyzing RNA diversity in eukaryotic transcriptomes: peering through the Omics lens. F1000Research. 2016;5:2668
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
References:
Berkovits B. D. and Mayr C. Alternative 3′ UTRs act as scaffolds to regulate membrane protein localization. Nature. 2015;522:363-367
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History: 3′-Seq
Revision by sbrumpton on 2017-06-21 07:50:23 - Show/Hide
Quantitatively Measure Abundance of 3'UTR Isoforms
3'-Seq was designed to measure the abundance of 3'-UTR isoforms quantitatively in a wide array of human tissue types (Lianoglou et al., 2013). 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.
Advantages:- Measures 3'UTR isoform abundance to detect protein expression differences in multiple tissue types
Disadvantages:- Technically challenging; nick translation requires precise conditions
Reagents:Illumina Library prep and Array Kit SelectorReviews:Bangru S. and Kalsotra A. Advances in analyzing RNA diversity in eukaryotic transcriptomes: peering through the Omics lens. F1000Research. 2016;5:2668Miura 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;36:766-777References:Berkovits B. D. and Mayr C. Alternative 3' UTRs act as scaffolds to regulate membrane protein localization. Nature. 2015;522:363-367