title: MCT1 causes resistance to SMAD3 inhibition in cancer and involves dysregulation of the cholesterol synthesis pathway (CRISPR-SAM gene activation screen)
design: MCT1 causes resistance to SMAD3 inhibition in cancer and involves dysregulation of the cholesterol synthesis pathway (CRISPR-SAM gene activation screen)
library_construction_protocol: 501Mel Naïve melanoma cells were engineered to stably express dCas9-VP64 (Addgene #61425) and MS2-P65-HSF1 (Addgene #61426) to enable gene activation by CRISPR-SAM. Lentivirus was produced in HEK293 cells using pPAX2 (Addgene #12259) and pMD2.G (Addgene #12259) vectors according to previously established protocols (D.Trono lab). Cells were incubated with lentivirus overnight in presence of 5μg/ml polybrene and selected during 5-10 days using Blasticidin (2-4μg/ml) or Hygromycin (200-400μg/ml). 501Mel (CTRL) 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. Human CRISPR lncRNA Activation Pooled Library (SAM 3-Plasmid System) was used for transcriptional activation of lncRNA genes (Addgene Pooled Library #1000000106). The library contains 96458 sgRNAs targeting 10504 genes (10 sgRNA per gene on average) as well additional 500 sgRNA controls. Lentiviral library was prepared according to previously established protocol (Joung et al., 2017, Nat Protoc) with minor modifications. Shortly, plasmid library was amplified in Lucigen E cloni. 10G Elite electrocompetent bacteria (Cat. #60051-2, LGC Biosearch technologies), grown on large squared LB agar plates overnight and purified using NucleoBond Xtra Maxi kit (Macherey-Nagel). To produce the lentiviral library, HEK293 cells were transfected 2 days before the infection of 501Mel CTRL cells. Media change was performed 3h before the lentiviral library infection (RPMI-1640 culture medium supplemented with 10% decomplemented FBS). 501Mel CTRL cells at 70-80% confluency were transduced with lentiviral library at MOI 0.2 in the presence of 5μg/ml polybrene. 250M cells were infected to maintain sufficient sgRNA coverage in cell library (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 500μg/ml zeocin for 10-14 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 500μg 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 350μg gDNA for each sample was used for PCR amplification of sgRNA target site sequences. For control, 10ng of pooled plasmid library was amplified. 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 lenti sgRNA(MS2)_zeo (Plasmid #61427) backbone used in the pooled library (Joung et al., 2017, Nature). 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 271-278bp 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.