TAIL-Seq focuses on sequencing the very ends of mRNA molecules (3'-UTRs and poly(A) tail regions) to explore their role in mRNA half-life, stability, their impact on translational efficiency, and to discover other aspects surrounding 3'-terminome function.
Ribosomal RNA (rRNA) is first removed from total RNA by affinity-based depletion. After purification, mRNAs are ligated to biotinylated 3' adapters prior to RNase T1 fragmentation and purified further by streptavidin pull-down. The 5' ends are phosphorylated and size-selected for 500–1000 nt fragments to prevent short noncoding RNA (ncRNA) fragments from contaminating the sequence data. The phosphorylated 5' ends are ligated to 5' adapters, reverse-transcribed, PCR-amplified, and sequenced. TAIL-Seq uses a unique paired-end run system to correlate the genes corresponding to the 3'-end sequence reads. By separating the paired-end reads, read 1 sequences 52 nt from the 5' end of the fragment to map the genome and identify transcripts, while read 2 sequences 251 nt from the 3' end specifically for sequence determination..