For pooled experiments, two Cas9-expressing HEK293T cell lines were used. The first expresses Cas9 from a piggybac integrated, TRE3G driven, doxycycline-inducible (1μg/ml) cassette, which we have previously described18 (link). The second expresses Cas9 constitutively from a CBh promoter in the AAVS1 Safe Harbor locus (GeneCopoeia #SL502). All HEK cells were cultured in DMEM +GlutaMax supplement (Thermofisher #10566016).
T25 cultures were transiently transfected with 12.76 ug of pooled plasmid per T25 using Lipofectamine 3000. Cultures were passaged for an additional 48 h. In the inducible Cas9 line, doxycycline was refreshed at passaging. Three days after transfection, cells were collected for sequencing analysis. To prepare samples for sequencing, cell pellets were collected, and gDNA was extracted using a QIAamp DNA mini kit according to the manufacturer’s instructions. DNA was eluted in 200 μl of ultra-pure, nuclease-free water.
T25 cultures were transiently transfected with 12.76 ug of pooled plasmid per T25 using Lipofectamine 3000. Cultures were passaged for an additional 48 h. In the inducible Cas9 line, doxycycline was refreshed at passaging. Three days after transfection, cells were collected for sequencing analysis. To prepare samples for sequencing, cell pellets were collected, and gDNA was extracted using a QIAamp DNA mini kit according to the manufacturer’s instructions. DNA was eluted in 200 μl of ultra-pure, nuclease-free water.
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