TITLE: Electroporation of hPSCs
AUTHORS: Hanqin  Li, Oriol Busquets, Steven Poser, Dirk Hockemeyer, Frank Soldner

[DESCRIPTION]
This protocol describes the standard procedure for the delivery of plasmids into human pluripotent stem cells (hPSCs) using electroporation.

Protocol Overview 
A. Preparation of MEFs-cultured hPSCs for electroporation
B. Preparing plasmids for electroporation
C. Electroporation

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.

[STEPS]
SECTION: A. Preparation of MEFs-cultured hPSCs for electroporation
1. Incubate the hPSC cultures (on MEF feeders) in hPSC medium containing 10 µM Y-27632 (1:1000 dilution of stock) for at least two hours (overnight works as well) before electroporation. Two to three near confluent 6-well plates should provide sufficient cells for each individual experiment (higher than 10X106 hPSCs/experiment) 

For a detailed protocol on hPSC cultures 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: A. Preparation of MEFs-cultured hPSCs for electroporation
2. Wash the hPSC plates twice with DPBS
SECTION: A. Preparation of MEFs-cultured hPSCs for electroporation
3. Add 1 ml Trypsin/well of a 6-well plate and incubate for 5 min  at 37 °C  until colonies start detaching from MEF feeders. 

SECTION: A. Preparation of MEFs-cultured hPSCs for electroporation
4. Add 2 ml hPSC medium to inactivate the trypsin.
SECTION: A. Preparation of MEFs-cultured hPSCs for electroporation
5. Collect single cell or small clump solution by trituration with P1000 pipette tips into 50 ml conical tube.
SECTION: A. Preparation of MEFs-cultured hPSCs for electroporation
6. Gravity precipitate cell solution for 5 min  min and remove large clumps of feeders with 5 ml pipette from the bottom of the conical tube. 

SECTION: A. Preparation of MEFs-cultured hPSCs for electroporation
7. Centrifuge at225 x g, 10 min 

SECTION: A. Preparation of MEFs-cultured hPSCs for electroporation
8. Remove the supernatant and re-suspend the cells at high concentration (higher than 10X106cells/700 µl) in ice-cold DPBS.
SECTION: B. Preparing plasmids for electroporation
9. Thaw plasmids on ice 

SECTION: B. Preparing plasmids for electroporation
10. Dependent on the specific experiment (ZNFs-, TALEN- or CRISPR/Cas9-based genome editing) we recommend keeping the overall amount of plasmid DNA for each experiment below 50 µg.
SECTION: B. Preparing plasmids for electroporation
11. For CRISPR/Cas9 genome editing, use:
    16 µg pX330-GFP gRNA (alternative: pX330-mcherry/BFP/YFP)
    34 µg ssODN (single strand oligonucleotide containing modification, ~100 bp)
SECTION: B. Preparing plasmids for electroporation
12. For TALEN genome editing, use 
    7.5 µg for each (left and right) TALEN-nuclease plasmid
    26 µg ssODN (single strand oligonucleotide containing modification, ~100 bp)
    10 µg pEGFP-N1 (or comparable fluorescent protein expression vector)
SECTION: B. Preparing plasmids for electroporation
13. For ZFN genome editing, use 
    7.5 µg for each (left and right) ZFN-nuclease plasmid
    26 µg ssODN (single strand oligonucleotide containing modification, ~100 bp)
    10 µg pEGFP-N1 (or comparable fluorescent protein expression vector)
SECTION: B. Preparing plasmids for electroporation
14. For prime editing PE2 strategy, use
    33 µg pCMV-PE2-GFP
    12 µg pU6-pegRNA
SECTION: B. Preparing plasmids for electroporation
15. For prime editing PE3 strategy, use
    33 µg pCMV-PE2-GFP
    12 µg pU6-pegRNA
    5 µg pBPK1520-ngRNA
SECTION: B. Preparing plasmids for electroporation
16. Pipet the proper amount of each plasmid into a micro centrifuge tube and keep on ice  (4°C) until electroporation. 

SECTION: C. Electroporation
17. Mix cell suspension (higher than 10X106cells/700 µl DPBS) with pre-made transfection solution (plasmid DNA), transfer in Biorad electroporation cuvette (4 mm) and incubate on ice for 10 min 

SECTION: C. Electroporation
18. Resuspend cell suspension cuvettes (either by careful vortexing or by hand) and pulse cuvettes using Biorad electroporator (Gene Pulser Xcell System, Bio-Rad: 250 V, 500 µF, 4 mm cuvettes). 
SECTION: C. Electroporation
19. Carefully collect the cells and transfer to pre-warmed hPSC medium containing 10 µM Y-27632 (1:1000 dilution of stock) and distribute electroporated cell solution on an entire 6-well MEF feeder plate (DR4-MEFs for selection-based experiments and ICR-MEFs for subsequent FACS sorting) 
SECTION: C. Electroporation
20. After plating shake the plate/s gently in the incubator and let it sit   (37°C; 5% CO2; 5% O2).

SECTION: C. Electroporation
21. Refresh hPSC medium daily until FACS or drug selection (usually after 48 – 72 hours). 
SECTION: A. Preparation of MEFs-cultured hPSCs for electroporation
1.1. hPSCs medium
     DMEM/F12      385ml        Fetal Bovine Serum (FBS)      75ml        Knockout Serum Replacement      25ml        L-Glutamine (100X)      5ml        Penicillin & Streptomycin (100X)      5ml        MEM Non-Essential Amino Acids (100X)      5ml        2-Mercaptoethanol (10,000X)      50µl        Heat Stable Recombinant Human FGF2 (25µg/ml)      80µl   
 
L-Glutamine (100X)
     L-Glutamine,   powder      14.6 g        MilliQ H2O      500 ml   
 
2-Mercaptoethanol (10,000X)
     2-Mercaptoethanol      0.78 ml        MilliQ H2O      9.22 ml   
 

Heat Stable Recombinant Human FGF2 (25µg/ml)
     Heat Stable Recombinant Human FGF2      500 ug        0.1% BSA      20 ml   
 
 
  Y-27632 (1,000X)
     Y-27632      5 mg        DMSO      1.56ml   
 
 
 hPSCs medium + Rock inhibitor
     hESCs medium      500ml        Y-27632 (1,000X)      500  µl