TL-Seq
Transcript Leader Sequencing
TL-Seq targets and enriches for the sequence around the 5′-UTRs of 5′-capped mRNA molecules before sequencing (Arribere et al., 2013). Poly(A)+ RNA is fragmented and selected for 50_80 nt fragments. RNA fragments with phosphorylated 5′ ends are dephosphorylated, using calf intestinal phosphatase (CIP), to distinguish them from capped mRNA fragments. Next, TAP strips the capped mRNAs and exposes the phosphate on the 5′ end for P5 adapter ligation. The adapter-ligated fragments are gel-purified by molecular weight before ligation of P7 adapters. The 3′-end adapters can be attached by poly(A) tailing and ligation, or directly if using preadenylated adapters. After RT and PCR amplification, the RNA library is sequenced.
Advantages:
- Sequences 5′ UTRs and identifies variants
- Able to associate transcript leader function in translation when combined with translation-associated TL-Seq (TATL-Seq)
Disadvantages:
- Selects short nucleotide fragments (50_80 nt)
- Labor-intensive; requires large amounts of starting material
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
Smith J. E. and Baker K. E. Nonsense-mediated RNA decay–a switch and dial for regulating gene expression. Bioessays. 2015;37:612-623
References:
Arribere J. A. and Gilbert W. V. Roles for transcript leaders in translation and mRNA decay revealed by transcript leader sequencing. Genome Res. 2013;23:977-987