Illumina HiSeq 4000 paired end sequencing; Single cell RNA sequencing of naive and primed human pluripotent stem cells treated with SB431542
Single cell RNA sequencing of naive and primed human pluripotent stem cells treated with SB431542
WA09/H9 NK2 naïve and WA09/H9 primed hESCs were grown in presence of 20µM (naïve) and 10µM (primed) of SB431542 for 7 days. Cells during the time-course were collected at day 0 (control, no SB) and at day 1, 3, 5 and 7 of treatment with Accutase (ThermoFisher Scientific) for 5min at 37˚C in hypoxia (5% O2), and resuspended until single cell suspension was obtained. Accutase was blocked by adding PBS/BSA 0.5% with the respective media, and after a wash pellets were resuspended at a concentration of ~1,000 cells/µl in the respective media. 3,000 cells/sample were loaded on a Chromium Chip B Single Cell following the manufacturer's instruction to generate Gel Beads-in-emulsion (GEMs) using a Reagent kit v3. Final Chromium Single Cell 3' Gene Expression library was generated using standard Illumina paired-end constructs with P5 and P7 primers using a Reagent kit v3.