Comprehensive determination of transcription start sites derived from all RNA polymerases using ReCappable-seq

We develop a method Re-Cappable-seq for determining eukaryotic transcription start sites derived from all RNA polymerases at nucleotide resolution. In particular, this method identifies the Pol-I and Pol-III TSSs, which are missing by CAGE. Applied to human A549 cell line, our method results in the identification of 33,468 and 5,269 high confidence Pol-II and non-Pol-II TSS respectively. Re-Cappable-seq identifies Pol-II TSS with higher specificity than CAGE. We isolated total RNA from human A549 cancer cell line. First we used yDcpS to decap the canonical G-cap structure from Pol-II transcripts leaving a di-phosphorylated 5'end. Then we used VCE to specifically add a biotin cap to these di-phosphorylated 5'ends as well as the tri-phosphorylated 5'ends from the non-Pol-II transcripts. Finally we isolated these biotin capped RNA using streptavidin beads and sequenced using illumina. Here the RNA without streptavidin enrichment served as control. In addition, we also performed CIP treatment before decapping, which was used to distinguish the Pol-II and non-Pol-II transcripts.