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.
- Measures 3’UTR isoform abundance to detect protein expression differences in multiple tissue types
- Technically challenging; nick translation requires precise conditions
Illumina Library prep and Array Kit Selector
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