Jan 18, 2026

Public workspaceGenomic engineering of iPSCs

DOI
https://dx.doi.org/10.17504/protocols.io.yxmvmbdzbg3p/v1
  • Ellen Hertz1,
  • Yu Chen1,
  • Joel Blanchard2,
  • Ellen Sidransky1
  • 1National Institutes of Health;
  • 2Icahn School of Medicine at Mount Sinai
Ellen Hertz
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DOI: https://dx.doi.org/10.17504/protocols.io.yxmvmbdzbg3p/v1
Protocol Citation: Ellen Hertz, Yu Chen, Joel Blanchard, Ellen Sidransky 2026. Genomic engineering of iPSCs. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvmbdzbg3p/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 11, 2025
Last Modified: January 18, 2026
Protocol Integer ID: 234760
Keywords: ASAPCRN, genomic engineering of ipsc, genomic engineering, ipsc
Funders Acknowledgements:
ASAP
Grant ID: ASAP-000458
Abstract
Genomic engineering of iPSCs as in Hertz et al 2025
Troubleshooting
Prepare cells for transfection
Grow iPSCs in Essential 8 Medium (Cat.# A1517001 Thermo Fisher) until ca 70% confluency
Wash cells with PBS
Dissociate iPSC into single cells with 1 ml StemPro-Accutase (Cat.# A1110501; Gibco)
Incubate at 37 C for 5 min
Resuspend cells in DMEM and spin down at 300 x g for 3 min
Resuspend cells in Essential 8 Medium with Y-27632 and seed at a density of 200,000 cells/cm2
Let cells settle for 2 hours at 37 degrees
Transfection
Mix Lipofectamine Stem Transfection Reagent (Cat.# STEM00001; Invitrogen) and plasmids in OPTI-MEM (Cat # 31985062, Thermo) according to instuctions
Incubate for 5 min at RT
Add drop wise to the cells
Incubate over night at 37 C and 5% CO
Selection of transfected cells
Reseed cells (as described above) and resuspend cells in StemFlex (Cat.# A3349401; Thermo Scientific), supplemented with Y-27632
Incubate 24 h
Start selection with 0.25 mg/ml puromycin (Cat.# A11138-03, Thermo Scientific)
Change media (including puromycin) every 2-3 days until the selected clones are almost confluent (days-weeks)
CRE-mediated excision of the selection cassette
Transfection of plasmid encoding GFP-tagged CRE-recombinase to remove the selection cassette as above. Incubate 24 h in cell incubator
Coat 96 well plate with laminin-521 overnight in incubator
FACS sort individual clones into laminin coated plate with Stemflex supplemented with 10 μM Y-27632, 5 μM Emricasan (Cat.# 7310, R&D), 1x penicillin-streptomycin, 0.7 μM ISRIB (Cat.# 5284, R&D) and  1x polyamines (Cat.# P8483, Sigma)
Incubate cells 3 days
Change 1/2 media with Stemflex supplemented with penicillin-streptomycin, ISRIB and polyamines
Incubate 3 days
Change 1/2 media with Stemflex every 3 days until clones reach ca 50% confluency.
Expand clones