Cell Culture and Lentivirus Production

RPMI-1640 with 25mM HEPES, 2.0 g/L NaHCO3, 0.3 g/L L-Glutamine supplemented with 10% FBS, 2 mM glutamine, 100 units/mL penicillin and 100 μg/mL streptomycin was used to grow K562 cells. HEK293T cells, used for packaging lentivirus, were grown in Dulbecco’s modified eagle medium (DMEM) in 10% FBS, 100 units/mL penicillin and 100 μg/mL streptomycin. Induced pluripotent stem cells (iPSCs) expressing Cas9 (WTC CRISPRn Gen1C^{27 (link)}) were maintained under feeder-free conditions on growth factor-reduced Matrigel (Corning) in mTeSR medium (STEMCELL Technologies). Accutase (STEMCELL Technologies) was used to enzymatically dissociate iPSCs into single cells to passage with 10 μM p160-Rho-associated coiled-coil kinase (ROCK) inhibitor Y-27632 (Selleckchem) added to promote cell survival. Lentivirus was produced by co-transfecting HEK293T cells with transfer plasmids and standard packaging vectors using TransIT®-LTI Transfection Reagent (Mirus, MIR 2306).

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Replogle J.M., Norman T.M., Xu A., Hussmann J.A., Chen J., Cogan J.Z., Meer E.J., Terry J.M., Riordan D.P., Srinivas N., Fiddes I.T., Arthur J.G., Alvarado L.J., Pfeiffer K.A., Mikkelsen T.S., Weissman J.S, & Adamson B. (2020). Combinatorial single-cell CRISPR screens by direct guide RNA capture and targeted sequencing. Nature biotechnology, 38(8), 954-961.

Publication 2020

growth factor-reduced Matrigel mTeSR medium Accutase p160-Rho-associated coiled-coil kinase (ROCK) inhibitor Y-27632 TransIT®-LTI Transfection Reagent

Accutase Cell survival Cells Eagle Glutamine Growth factor Hepes Induced pluripotent stem cells K562 cells Lentivirus Matrigel Nahco3 P160 Penicillin Plasmids Rho associated coiled coil kinase Stemcell Streptomycin Transfection Vectors Y 27632

Corresponding Organization :

Other organizations : University of California, San Francisco, 10X Genomics (United States), Howard Hughes Medical Institute, Princeton University

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Variable analysis

independent variables

Growth medium used to culture K562 cells (RPMI-1640 with 25mM HEPES, 2.0 g/L NaHCO3, 0.3 g/L L-Glutamine supplemented with 10% FBS, 2 mM glutamine, 100 units/mL penicillin and 100 μg/mL streptomycin)
Growth medium used to culture HEK293T cells (Dulbecco's modified eagle medium (DMEM) in 10% FBS, 100 units/mL penicillin and 100 μg/mL streptomycin)
Growth medium used to culture induced pluripotent stem cells (iPSCs) expressing Cas9 (mTeSR medium)
Dissociation method used for passaging iPSCs (Accutase)
ROCK inhibitor Y-27632 used to promote iPSC survival during passaging
Transfection method used to produce lentivirus (co-transfection of HEK293T cells with transfer plasmids and standard packaging vectors using TransIT®-LTI Transfection Reagent)

dependent variables

Growth and proliferation of K562 cells
Growth and proliferation of HEK293T cells
Maintenance and passage of iPSCs expressing Cas9
Lentivirus production

control variables

Concentration of HEPES, NaHCO3, L-Glutamine, FBS, penicillin, and streptomycin in the growth media
Concentration of FBS, penicillin, and streptomycin in the HEK293T growth medium
Growth factor-reduced Matrigel used as the substrate for iPSC culture
Concentration of ROCK inhibitor Y-27632 used during iPSC passaging

positive controls

None specified

negative controls

None specified

Annotations

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