GSM8781767: DMSO control for TM samples, 2; Homo sapiens; OTHER
Five 15 cm (diameter) dishes of 60 % confluent cells per condition were put on ice, washed with ice-cold PBS (Sigma) and crosslinked with the UV light at 365nm with 0.15 J/cm2 and pelleted. Cell pellets were lysed in 1250 μL of ice-cold lysis buffer (25 mM HEPES pH 7.3, 150 mM NaCl, 0.5% NP-40, 0.5 mM EDTA, 10% Glycerol, 0.1% SDS, 0.2% Sodium Deoxycholate, 1x protease inhibitors cocktail, 0.1mM DTT), by incubation on ice for 15 min with intermittent vortexing and passing three times through the 27G needle. The lysate was clarified by centrifugation for 20 min at 20,000g at +4ºC. An aliquot of the lysate was taken before the in-lysate RNase digest for total RNA isolation and sequencing. Total RNA was isolated using peqGOLD TriFast (Peqlab, VWR). Transcriptome libraries were prepared with QuantSeq 3' mRNA-Seq Library Prep Kit (Lexogen). Libraries were sequenced on a NextSeq2000 P2 at SR100 mode (Illumina) at the Vienna BioCenter NGS facility with 30 million reads per sample on average.