GSM8679297: DMSO_Nuc_3; Homo sapiens; RNA-Seq
Cells were trypsinised, washed with cold PBS, counted, and Drosophila S2 cells spiked in prior to permeabilisation. Cells were permeabilised (1mg/ml Digitonin (SKU0215948050, MP Biomedicals), 100mM NaCl, 10mM Tris pH 8.0, 1:100 protease Inhibitor (Complete Mini EDTA-free, 4693159001 Sigma), 1:1000 RNase OUT (10-777-019, Invitrogen) for 10 min on ice. After centrifugation at 1000g for 3 min, the supernatant (cytoplasmic fraction) was set aside and the pelleted nuclei were resuspended in RLT buffer (Qiagen) and put through a QIAshredder column (Qiagen). Both cytoplasmic and nuclear fractions were then diluted 1:3 with RLT buffer (Qiagen), and ethanol added to a final concentration of 30%. RNA was recovered using a RNeasy mini spin column (Qiagen).carboxyamidomethylation of 4sU was performed using freshly prepared iodacetamide as described (Herzog et al Nature Methods 2017 PMID: 28945705). ERCC RNA (4456740, Ambion; 2l per 2g RNA) was spiked in according the manufacturer's instructions. RNA was cleaned up using MinElute RNeasy kit (Qiagen). Libraries were prepared using NEBNext Ultra II RNA Library Prep Kit for Illumina with poly-A selection (NEB). Libraries were sequenced on a Novaseq with 150bp paired-end reads.