Genome-Wide Mapping of 5' Isoforms with 5'-Seq

Transcriptome of a genome is appreciated to be more complex than previously assumed. Same gene readouts can differ in terms of transcription start site, transcription end site and splicing. Growing evidence suggests functional importance of distinct transcript isoforms of the same gene. Obtaining these isoforms easily experimentally and processing data is crucial for prompt transcriptome functional characterizations. Here, I describe a quick protocol for generation of capped 5 isoforms sequencing library and 5 isoforms data analysis. The protocol relies on utilization of dephosphorylation-decapping method (oligo-capping), and it is a simplification of previously published 5 isoform studies. The pipeline for data analysis suggests several isoform features to focus on.