TITLE: FACS-based enrichment of transfected hPSCs
AUTHORS: Hanqin  Li, Oriol Busquets, Steven Poser, Dirk Hockemeyer, Frank Soldner

[DESCRIPTION]
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.

[STEPS]
SECTION: A. Preparation of samples for FACS sorting
1. Incubate the hPSC cultures (either feeder-free in mTeSR media or on MEF feeders in hPSC medium) supplemented with 10 µM Y-27632 (1:1000 dilution of stock) for at least .120 min 

For a detailed protocol on growth of hPSCs in feeder-free media refer to the collection "Feeder-free culturing of hPSCs;" dx.doi.org/10.17504/protocols.io.b4mcqu2w

For a detailed protocol on growth of hPSCs on feeders, refer to the collection "Maintenance and inactivation of mouse embryonic fibroblasts (MEFs) as feeder cells for human pluripotent stem cell culture;" dx.doi.org/10.17504/protocols.io.b4pbqvin
SECTION: A. Preparation of samples for FACS sorting
2. Wash the plates twice with DPBS
SECTION: A. Preparation of samples for FACS sorting
3. Add 1 ml of 0.05% Trypsin and incubate for 5 min 37 °C 

SECTION: A. Preparation of samples for FACS sorting
4. Add 2 ml of hPSC medium or trypsin inhibitor solution per well of a 6-well plate to inhibit trypsin
SECTION: A. Preparation of samples for FACS sorting
5. Collect single cell solution by careful trituration with P1000.
SECTION: A. Preparation of samples for FACS sorting
6. Filter cell solution through cell strainer (70 µm) into 50 ml conical tube.
SECTION: A. Preparation of samples for FACS sorting
7. Centrifuge at 225 x g, 10 min  

SECTION: A. Preparation of samples for FACS sorting
8. Remove the supernatant and re-suspend the cells in 0.5 ml hPSC medium or feeder-free medium with 10 µM Y-27632 (1:1000 dilution of stock).
SECTION: A. Preparation of samples for FACS sorting
9. Transfer and filter re-suspended single cell solution in 5 ml FACS tubes with cell strainer snap Cap (40 µm)
SECTION: A. Preparation of samples for FACS sorting
10. Substitute filter caps for completely closed ones (maintain sterile conditions) and place the tubes on ice.
SECTION: A. Preparation of samples for FACS sorting
11. Provide FACS core facility with cells, negative controls, collection tubes (containing 1 ml of hPSC medium or feeder-free medium with 10 µM Y-27632 (1:1000 dilution of stock)) and detailed experimental information to enable the setup of appropriate FACS parameters.
SECTION: B. After FACS
12. Clonal expansion:
The FACS-sorted (transfected) cells can be plated at low density on MEF feeders (approx. 2000-3000 cells/well of a 6-well plate) in hPSC medium containing 10 µM Y-27632 (1:1000 dilution of stock) (only for the first 24 hours after plating). This will allow for identification individual single cell derived colonies 10-14 days after plating.

For a detailed protocol on maintenance of hPSCs on MEF feeders, refer to the collection "Maintenance and inactivation of mouse embryonic fibroblasts (MEFs) as feeder cells for human pluripotent stem cell culture;" dx.doi.org/10.17504/protocols.io.b4pbqvin

SECTION: B. After FACS
13. Bulk genotyping:
The FACS-sorted (transfected) cells can be directly subjected to DNA extraction and next-generation sequencing (NGS). Depending on the cell number (small cell number) for DNA-extraction, we usually use Exact N Amp Blood PCR Kit (Sigma) according to the manufacturers’ instructions. 

For a detailed protocol on NGS genotyping refer to the collection "Genotyping by next generation sequencing;" dx.doi.org/10.17504/protocols.io.b4n3qvgn