Simultaneous Measurement of Transcriptional and Post-transcriptional Parameters by 3' end RNA-seq

Cellular RNA levels are determined by transcription and decay rates, which are fundamental in understanding gene expression regulation. Measurement of these two parameters is usually performed independently, complicating analysis and introducing methodological biases that hamper direct comparison. Here, we present a simple approach of concurrent sequencing of S. cerevisiae polyA+ and polyA- RNA 3' ends to simultaneously estimate total RNA levels, transcription and decay rates from the same RNA sample. The transcription data generated correlate well with reported estimates and also reveal local RNA polymerase stalling and termination sites with high precision. Although the method by design uses brief metabolic labeling of newly synthesized RNA with 4-thiouridine, the results demonstrate that transcription estimates can also be gained from unlabeled RNA samples. These findings underscore the potential of the approach, which should be generally applicable to study a range of biological questions in diverse organisms. RNA 3' end seq of total and 2min 4-thiouracil (4tU) labelled RNA from S. cerevisiae cells. Aliquots of RNA were directly subjected to pA+ RNA 3' end sequencing (noPap samples). A second aliquot was in vitro polyadenylated using E. coli poly(A) polymerase and ribodepleted before library preparation (xPap samples).