GSM7430319: sc_his-0_rep3; Saccharomyces cerevisiae; OTHER
Total RNA was isolated by the standard phenol chloroform method and DNA was removed by DNase I treatment, as described in Zhang & Pelechano, 2021 (PMID:33870233). RNA concentration was measured with Qubit and RNA quality was checked by 1.2% Agarose Gel or on a BioAnalyzer using an RNA Nano 6000 chip according (Agilent Technologies). HT-5Pseq libraries were prepared following a previously established protocol (Zhang & Pelechano, 2021) if not stated otherwise. To briefly summarize, 6 μg of RNA was subjected to DNase treatment, and the resulting DNA-free total RNA was ligated with an RNA/RNA oligo containing UMI (RNA rP5_RND oligo). The ligated RNA was reverse transcribed using Illumina PE2 compatible oligos with random hexamers and oligo-dT as primers. To eliminate RNA in RNA/DNA hybrids, samples were treated with sodium hydroxide at 65°C for 20 minutes. Ribosomal RNAs were depleted using DSN (Duplex-specific nuclease) and a mixture of ribosomal DNA probes. To deplete Ribosomal RNAs in B. subtilis 168 (trpC2), we used the in-house rRNA DNA oligo depletion mixes described previously (Huch, Nersisyan et.al, doi.org/10.1101/2021.04.08.439066). Finally, the samples were amplified by PCR and sequenced on an Illumina NextSeq 2000 instrument using average of 45 cycles for read1.