5c48bf3a-9048-458b-9503-bd101ad3353c
A map of metabolism in the DNA damage response identifies PRDX1 in nuclear ROS scavenging and aspartate synthesis
Library amplification: The Sabatini metabolic CRISPR pooled library was amplified following the distributor's instructions (Addgene), with a coverage of around 200X.  Virus production: HEK-xLentiTM cells (Oxgene) were seeded in 12xT225 flasks 10cm dishes and transfected 24 hours later, with the metabolic CRISPR pooled library, pVSVG and psPAX2 packaging plasmids, using polyethylenimine (PEI) in OptiMeM (Gibco). Medium was changed 10 hours later. 24 and 48 hours later, supernatant containing virus was harvested and centrifuged at 2000 rpm for 5 minutes to remove cell debris. The two batches were pooled together and the virus was concentrated 20X using PEG-8000 and stored at -80°C.  Cell infection and harvest: U2OS cells were spinfected for 3 hours at 2000 rpm and 37 degrees in 12-well plates with the lentiviral metabolic library at a multiplicity of infection (MOI) of 0.3-0.5 in presence of polybrene (final concentration 8 ug/mL). Immediately after spinfection, cells were collected and seeded in 245mm square dishes with fresh medium. Puromycin-containing medium (1.5 ug/ml) was added the next day to select for transductants. At 7 days post-transduction, cells were re-seeded, and at 9 days post-transduction, they were either treated with 1µM etoposide for 3 hours or left untreated. Treated cells were washed with PBS and released in drug-free media, and after 24 hours release, both untreated and treated cells were harvested. Part of the harvested cells were re-seeded to be harvested at a later timepoint, maintaining 1000X coverage, while the rest of the cells were fixed with ice cold 90% methanol in PBS at a density of 8 million cells/mL, and stored in methanol at -20°C. At 14 days after transduction, both untreated and treated cells were harvested, methanol-fixed and stored at -20°C.
Library amplification: The Sabatini metabolic CRISPR pooled library was amplified following the distributor's instructions (Addgene), with a coverage of around 200X.  Virus production: HEK-xLentiTM cells (Oxgene) were seeded in 12xT225 flasks 10cm dishes and transfected 24 hours later, with the metabolic CRISPR pooled library, pVSVG and psPAX2 packaging plasmids, using polyethylenimine (PEI) in OptiMeM (Gibco). Medium was changed 10 hours later. 24 and 48 hours later, supernatant containing virus was harvested and centrifuged at 2000 rpm for 5 minutes to remove cell debris. The two batches were pooled together and the virus was concentrated 20X using PEG-8000 and stored at -80°C.  Cell infection and harvest: U2OS cells were spinfected for 3 hours at 2000 rpm and 37 degrees in 12-well plates with the lentiviral metabolic library at a multiplicity of infection (MOI) of 0.3-0.5 in presence of polybrene (final concentration 8 ug/mL). Immediately after spinfection, cells were collected and seeded in 245mm square dishes with fresh medium. Puromycin-containing medium (1.5 ug/ml) was added the next day to select for transductants. At 7 days post-transduction, cells were re-seeded, and at 9 days post-transduction, they were either treated with 1µM etoposide for 3 hours or left untreated. Treated cells were washed with PBS and released in drug-free media, and after 24 hours release, both untreated and treated cells were harvested. Part of the harvested cells were re-seeded to be harvested at a later timepoint, maintaining 1000X coverage, while the rest of the cells were fixed with ice cold 90% methanol in PBS at a density of 8 million cells/mL, and stored in methanol at -20°C. At 14 days after transduction, both untreated and treated cells were harvested, methanol-fixed and stored at -20°C.