The open reading frames of INPPL1 (GenScript: OHu19348), MAP4K5 (GenScript: OHu02673), and various mutants of these genes were cloned into pLV-EF1a-IRES-Neo (85139; Addgene) using a Gibson Assembly kit (E5510S; NEB). All sgRNAs were cloned into lentiCRISPRv2 (52961; Addgene) by following a published protocol (Sanjana et al., 2014 (link); Shalem et al., 2014 (link)). Lentiviruses were produced by co-transfecting HEK293T cells with viral construct and packaging vector DNAs (Hart et al., 2017 (link); Mair et al., 2019 (link)). Cells were spin-infected with concentrated viruses and infected cells were recovered by selection with the appropriate antibiotics.
Protocol detail
Cloning and Lentiviral Transduction of INPPL1 and MAP4K5
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Corresponding Organization : NYU Langone Health
Other organizations : New York University, New York Genome Center
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Variable analysis
independent variables
- INPPL1 (GenScript: OHu19348)
- MAP4K5 (GenScript: OHu02673)
- Various mutants of INPPL1 and MAP4K5
- Not explicitly mentioned
- Not explicitly mentioned
- Positive control: Not specified
- Negative control: Not specified
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