GSM8907784: W303 Prp19-AID DMSO-treated cells replicate 1; Saccharomyces cerevisiae; OTHER
Cell lysis and affinity purification of nascent 4-thiouracil labelled RNA was performed as described in O-Brien and Ansari (2023). The nascent, purified RNA samples were depleted of rRNA. GRO-seq libraries were constructed from the rRNA-depleted samples following the NEBNext Ultra II Directional RNA Library protocol. cDNA library quality was assessed to verify the library size (200-700 bp peak), purity and quantity using Agilent Tape Station 2200 gDNA-QG protocol following the manufacturer's protocol. Sequencing was performed on a NovaSeq 6000 (2 x 50bp, 10 M reads per sample). To perform sequencing of libraries using Illumina Sequencing system, libraries were normalized to a 1.5 nM pool for a final loading concentration of 300 pM on a NovaSeq 6000 following the Denature and Dilute Libraries Guide. GRO-seq reads were trimmed for quality before alignment to the Saccharomyces genome (Saccer3, downloaded from UCSC database) using hisat2 (version 2.1.0) with the rna-strandness parameter.