CRISPR-KO cell line (hES cells)

Passage 29 human WA01 H1 hESC were grown in mTeSR1 (Stem Cell #05850) feeder free system on matrigel (Corning 356230) coated plates. For transfection, cells were incubated with Thiazovivin (Santa cruz sc-361380) at 2 uM for 1 hour. Cells were treated with Accutase to single cell suspension and then pelleted and resuspended in DNA/lipofectamine solution and incubated at RT for 10–15 minutes. Cells were replated in thiazovivin-containing media on matrigel. At 48 hours post-transfection, cells were FACs sorted based on GFP expression and selected colonies were expanded for screening by immunoblotting.