TITLE: Subcloning of genotype-confirmed hPSCs clones
AUTHORS: Hanqin  Li, Yogendra Verma, Dirk Hockemeyer, Frank Soldner

[DESCRIPTION]
This protocol describes a standard procedure for subcloning of genotype-confirmed human pluripotent stem cells (hPSCs).

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]
1. Change medium to hPSCs medium + Rock inhibitor one day before subcloning 
2. Wash the genotype-confirmed wells with DPBS
3. Add 25 µl Trypsin to those wells
4. Incubate  5 min 37 °C 

5. Add 75 µl hPSCs medium + Rock inhibitor, mix well by pipetting
6. Transfer dissociated cells to a 15 conical tube
7. Prepare three MEF wells of a 6-well plate. Seed 1/100, 1/1,1000 and the rest of the cells respectively to those wells
8. Change medium daily for the high density well starting from day 3. Change medium every 3 days for the low density wells in the first week, then every other day in the second week
9. Once the high density wells grow to 50-70% confluency, freeze. 

For a detailed protocol on freezing hPSCs, refer to "Freezing of hPSCs grown on MEFs" in the "Thawing, Passaging and Freezing of hPSCs on MEFs" collection; dx.doi.org/10.17504/protocols.io.b4msqu6e

For a detailed protocol on freezing feeder-free hPSCs, refer to "Freezing of feeder-free hPSCs" in the "Feeder-free culturing of hPSCs" collection; dx.doi.org/10.17504/protocols.io.b4mcqu2w
10. When big hPSCs colonies form in the low density wells, change medium to hPSCs medium + Rock inhibitor
11. The day after, proceed with manual colony picking
12. For each original clone, prepare six microcentrifuge tubes pre-added with 20 µl Trypsin
13. Aspirate medium and add 2 ml DMEM/F12
14. Change medium to DPBS for the well where colonies will be picked 
15. Under the dissecting microscope, use mouth pipet or a fine 10 µl tip to pick one undifferentiated colony which is fully separated from other colonies. 
16. Transfer the colony to one microcentrifuge tube from step 12. 
17. Repeat step 15-16 to pick another five colonies
18. Incubate the microcentrifuge tubes at 37 °C 5 min 

19. Add 70 µl hPSCs medium + Rock inhibitor to each tube, pipet to mix
20. Seed all cells into one well of a 12-well MEF plate
21. Shake plates to distribute cells evenly
22. Culture for 7-10 days with medium change daily from day 3
23. When the subclones grow to 50-70% confluence, passage and prepare crude cell lysis for NGS genotyping
24. Once genotype confirmed, expand and freeze the one subclone, which shows the best hPSCs morphology and proliferates normally. 
25. Test for mycroplasma, stain for pluripotent markers, and karyotyping 


1.1. hPSC medium
     DMEM/F12      385 ml        Fetal   Bovine Serum (FBS)      75 ml        Knockout Serum   Replacement      25 ml        L-Glutamine (100X)      5 ml        Penicillin &   Streptomycin (100X)      5 ml        MEM Non-Essential Amino   Acids (100X)      5 ml        2-Mercaptoethanol   (10,000X)      50 ul        Heat Stable Recombinant   Human FGF2 (25ug/ml)*      80 ul   
 
 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      1.56 ml   
 

hPSC medium + Rock inhibitor
     hESCs medium      500 ml        Y-27632   (1,000X)      500 ul   

23.1. Crude lysis buffer (2x)