title: MCT1 causes resistance to SMAD3 inhibition in cancer and involves dysregulation of the cholesterol synthesis pathway (CRISPR-KO screen)
design: MCT1 causes resistance to SMAD3 inhibition in cancer and involves dysregulation of the cholesterol synthesis pathway (CRISPR-KO screen)
library_construction_protocol: 501Mel Naïve melanoma cells were maintained at 37°C and 5% CO2 in humidified air and grown in RPMI-1640 culture medium supplemented with 10% FBS and 1% penicillin–streptomycin. Cells were grown in T175 cell culture flasks. Media change was performed 3h before the lentiviral library infection (RPMI-1640 culture medium supplemented with 10% decomplemented FBS). Human CRISPR Knockout Pooled Library (Brunello) was used for CRISPR-Cas9 KO screen to target exons of protein-coding genes (Addgene Pooled Library #73179-LV). The library contains 76441 sgRNAs targeting 19114 genes (4 sgRNA per gene on average) as well additional 1000 sgRNA controls. 501Mel Naïve (parental) cells at 70-80% confluency were transduced with lentiviral library at MOI 0.5 in the presence of 5μg/ml polybrene. 100M cells were infected to maintain sufficient sgRNA coverage in cell library (at least 500 cells per each sgRNA). Cells were incubated with lentivirus for 24h, followed by media change containing RPMI-1640 supplemented with 10% FBS and 1% penicillin–streptomycin and incubated for additional 24h. Cells were then treated with 2.5μg/ml puromycin for 2-3 days for selection of infected cells. After cell library expansion, 150M cells were collected for each of 3 replicates of non-treated control condition and frozen. Remaining cells were seeded in 3 replicates (150M cells for each replicate) and treated with 17μM SIS3 over 2 weeks. Cells were split with 2/5 ratio after every 2-3 days at 90% confluency. After 2 weeks, 150M cells were collected for each of 3 replicates and frozen. All steps of growth protocol, treatment protocol and sample collection protocol were performed within the Inserm U1242 Unit. Frozen pellets of non-treated and SIS3-treated cells (150M cells for each replicate) were subjected to genomic DNA extraction using NucleoBond HMW DNA extraction kit (Macherey-Nagel). Cells were thawed on ice and dissolved in lysis buffer, followed by cell disruption through 25G needle (3 times) and overnight incubation at 50oC in presence of proteinase K. Genomic DNA was extracted according to the manufacturer's protocol and at least 600μg of gDNA was recovered for each replicate sample.  Nucleic acid library construction was performed according to Joung et al., (2017, Nat Protoc) with minor modifications. A total of 280μg gDNA for each sample was used for PCR amplification of sgRNA target site sequences. PCR reactions were performed using high-fidelity Herculase II Fusion DNA Polymerase (Agilent). sgRNA target site sequences were amplified using Illumina P5 and P7 NGS sequencing primers with binding sites targeting the lentiCRISPR v2 (Addgene Plasmid #52961) backbone used in the pooled library (Joung et al., 2017, Nat Protoc). A mixture of 8 P5 primers (different by stagger sequence length) and 1 P7 primer with unique barcode sequence were used for PCR reactions for each replicate sample. Size selection and cleanup steps of PCR product were performed using AMPure XP beads (Beckman Coulter) and purified DNA was eluted in H2O. Fragment size analysis was performed using TapeStation 4200 system (Agilent). Pure samples containing 260-267bp amplicons were pooled together with equimolar concentrations, re-purified and eluted in H2O. All steps of nucleic acid library construction protocol were performed within the Inserm U1242 Unit.