GSM9081933: PBMCs, treated with vehicle control and labelled with 4sU; Homo sapiens; RNA-Seq
The methanol fixation was allowed to proceed for 30 minutes at -20 °C. Then, 10 mM iodoacetamide (IAA) was added to each of the methanol-fixed cells, and the mixture was left at 4 °C overnight with gentle agitation. The next day, cells were centrifuged down, resuspended in quenching buffer containing DTT (DBPS, 0.1% BSA, 1 U/μl RNaseOUT, 100 mM DTT) for 5 min at room temperature. Cells were then centrifuged down, resuspended in resuspension buffer (DBPS, 0.01% BSA, 0,5 U/μl RNaseOUT, 1mM DTT). Cells were then passed through a 0,4m filter, counted, and proceeded for the 10X Genomics microfluidics run. PBMCs with and without K21 were loaded separately into a lane on the same chip, targeting 5000 cells per lane. The scRNA-seq libraries were constructed using the Chromium Single Cell 3' v3.1 Reagent Kit according to the manufacturer's guidelines (10× Genomics). cDNA libraries were uniquely sample-indexed and pooled for sequencing. A Illumina sequencing run was used to sample balance on a NextSeq 500 flowcell (Illumina) targeting > 50,000 reads/cell.