TL-Seq

TL-Seq

TL-Seq targets and enriches for the sequence around the 5'-UTRs of 5'-capped mRNA molecules before sequencing. 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.

Pros:

• Sequences 5' UTRs and identifies variants
• Able to associate transcript leader function in translation when combined with translation-associated TL-Seq (TATL-Seq)

Cons:

• Selects short nucleotide fragments (50–80 nt)
• Labor-intensive; requires large amounts of starting material

• TL-Seq: Arribere J. A. and Gilbert W. V. (2013) Roles for transcript leaders in translation and mRNA decay revealed by transcript leader sequencing. Genome Res 23: 977-987

1. Bangru S. and Kalsotra A. Advances in analyzing RNA diversity in eukaryotic transcriptomes: peering through the Omics lens. F1000Research. 2016;5:2668
2. Smith J. E. and Baker K. E. Nonsense-mediated RNA decay--a switch and dial for regulating gene expression. Bioessays. 2015;37:612-623

1. 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