GSM4910509: Control Untreated TT-seq replicate 3; Saccharomyces cerevisiae; RNA-Seq
Cell pellets were washed once with sterile water and snap-frozen in liquid nitrogen. Total RNA was extracted using the hot acid phenol protocol (acid phenol:chloroform:isoamyl alcohol 125:24:1) and TE-SDS buffer, then precipitated in ethanol with 0.3 M sodium acetate before re-suspension in DEPC-treated sterile water. Total RNA from yeast was cleaned up through column purification through the RNA Cleanup protocol within the RNeasy Mini Kit including DNase digestion (Qiagen). To measure global transcription levels a 4-TU pulsed S. pombe Total RNA was used as a spike-in for all samples, 90μg of sample Total RNA was combined with 10μg of S. pombe Total RNA. Nascent RNA from the samples containing also S. pombe spike-in RNA was isolated using TTchem-seq, this involved biotinylation of chemically fragmented Total RNA, the biotiylated nascent RNA was isolated from the fragmented Total RNA using a bead-based streptavidin pull down. 20 ng of nascent RNA was used to make libraries with the KAPA RNA HyperPrep Kit (Roche). The library was made following the manufacturer's instructions, with adjustments to ensure small transcripts from the fragmentation were not lost. The ratio of KAPA pure beads to adapter-ligated cDNA was 0.95x in the 1st post-ligation clean up, and 1x in the 2nd post-ligation clean up. Libary quailty was assessed using a Biolanalyzer 2100 (Agilent) and nascent RNA sequencing was carried out on the Illumina HiSeq 4000 platform and typically generated approximately 19 million 76 bp strand- specific single-end reads. library strategy: TT-seq