Neb 5 alpha electrocompetent e coli

The Perturb‐seq GBC library was a gift from Jonathan Weissman (Addgene ID #85968) (Adamson et al, 2016 (link)). The library was expanded in NEB® 5‐alpha electrocompetent E. coli (New England Biolabs, Ipswich, MA) according to the manufacturer's protocol, ensuring at least a 100× coverage to maintain library diversity. To produce lentivirus, library plasmids were co‐transfected with plasmids pLP1, pLP2, and VSVg (5 μg/ml of each, Thermo Fisher Scientific, Boston, MA) using PEI (100 μg/ml, PolySciences, Warrington, PA) into HEK293T packaging cells. Virus supernatant was collected at 48 and 72 h after transfection, and virus was purified by ultracentrifugation at 72,000 g for 2 h, for 2 h, re‐suspended in DMEM/F12 (Thermo Fisher Scientific), and stored in aliquots at −80°C until use.

PCR-amplification of the entire plasmid containing the gene of interest was performed with Phusion DNA polymerase (Thermo Fisher) using two complementary oligonucleotides containing a stretch of overlapping bases with the desired mutation in the middle (Supplementary file 1g). The reaction product was purified using GeneJET Gel Extraction Kit (Thermo Fisher) and digested with DpnI (Thermo Fisher) for 1 hr at 37°C to remove the template DNA. After digestion, the PCR product was purified and transformed into NEB 5-alpha electrocompetent E. coli (New England Biolabs) as described above, plated on LA plates containing 50 µg/ml of ampicillin, and incubated overnight at 37°C. Transformants were inoculated into fresh LB medium supplemented with 50 µg/ml of ampicillin and grown overnight at 37°C with shaking at 200 rpm. Plasmids were purified from the overnight cultures using the EZNA Plasmid Mini Kit (Omega Bio-Tek) according to the manufacturer’s protocol. Presence of the desired nucleotide substitutions were confirmed by Sanger sequencing.