Development of High-throughput 5Pseq and detection of ribosome stalls at termination level in Saccharomyces cerevisiae
We developed a high throughput 5'P sequencing approach (HT-5PSeq) to investigate 5'P mRNA degradome in relation with translation process.  This approach is easy and scalable; and uses an affordable rRNA depletion based on duplex-specific nuclease treatment. We use it to investigate in vivo ribosome stalls in S. cerevisiae and S. pombe at single nucleotide resolution. We investigate the 5'P degradation profiles associated to ribosome pausing, its regulation in stress conditions and the relative poly(A) length of mRNA degradation intermediates. Overall design: We performed 2 biologically independent HT-5PSeq experiments. For S.cerevisiae, we performed HT-5Pseq with DSN treatment with probes targeting ribosomal rRNA (BY-DSN) and none DSN treatment (BY-NonDSN) , DSN treatment without probes (BY-NP) as control. We also performed 5Pseq (PMID: 26046441) in S.cerevisiae as positive control. For S.pombe, we performed HT-5Pseq with DSN treatment with probes (orginally designed for S.cerevisiae ribosomal rRNA) (Pombe-DSN) and none DSN treatment (Pombe-NonDSN) as control.To inhibit translation process, we used cycloheximide (CHX) as a translation inhibitor on S.cerevisiae (BY-CHX) and S.pombe (Pombe-CHX). In addition, we performed HT-5Pseq under different growth conditions. For early exponential phase, strains were grown to a final OD600 of 0.3 (BY-lowOD). To reach stationary stage, strains were grown during 60h in YPD (BY-Stationary). We performed paired-end sequencing for read1 (61bp) and read2 (15bp) in some samples (see below). We splited libraries with polyA and non-polyA based on the length of As in read2. For other samples, we performed single end sequencing.