title: Illumina HiSeq 4000 paired end sequencing; Single cell RNA sequencing of naive and primed human pluripotent stem cells treated with SB431542
library_construction_protocol: 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.