Transient transcriptome sequencing: computational pipeline to quantify genome-wide RNA kinetic parameters and transcriptional enhancer activity

Abstract

In the accompanying chapter (Gressel, Lidschreiber, Cramer), we describe the detailed experimental protocol for transient transcriptome sequencing (TT-seq). TT-seq detects metabolically labeled, newly synthesized RNA fragments genome-wide in living cells. TT-seq can monitor gene activity and the dynamics of enhancer landscapes with great sensitivity, but this requires careful computational analysis of the data. In this manuscript, we present the bioinformatics workflow used to analyze TT-seq data. In particular, we describe pre-processing steps, including a reliable and robust normalization strategy, and several downstream analysis tools that enable the user to quantify RNA synthesis, splicing and degradation activities. Together, these tools form a comprehensive analysis pipeline that can be adapted to almost any TT-seq application.