FACS-based enrichment of transfected hPSCs v1

This protocol describes the procedure FACS-based enrichment of transfected human pluripotent stem cells (hPSCs). Protocol overview A. Preparation of samples for FACS sorting B. After FACS General notes 1. Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs. 2. Dependent on the genome editing strategy, we use either drug selection or FACS sorting to enrich transfected cell populations based on the presence of a fluorescent protein (either as part of the transfected plasmids or through co-transfection of a fluorescent protein expressing plasmid). 3. Either drug selection or FACS sorting will be usually performed 48 – 72 hours after electroporation/nucleofection. 4. Carefully plan the timeline of FACS experiments and schedule required FACS sorting time at core facility (requires approximately 45 min sorting time per sample) 5. Every FACS sorting experiment requires an additional non-electroporated well of hPSCs (parental cell line) as a negative control to determine the appropriate FACS gates.