GSM9684265: K562 dCas9-KRAB cells, hCRISPRi-v2 library, non-targeting sgRNA (sgNT), pre-treatment (T0), biological replicate 2; Homo sapiens; OTHER
Cell pellets from the screen were processed using the NucleoSpin Blood XL Kit (Macherey Nagel, 740950) according to manufacturer's directions and extracted DNA was quantified with DeNovix Nanodrop. After DNA extraction, samples were processed for NGS sequencing using the protocol described in Heo, SJ., Enriquez, L.D, Federman, S. et al51. DNA samples were amplified by PCR using NEBNext Ultra II Q5 MasterMix (NEB, M0544) with a common reverse primer (CAAGCAGAAGACGGCATACGAGATATGCTGTTTCCAGCTTAGCTCTT) and a unique forward primer per sample containing a 6 bp barcode (AATGATACGGCGACCACCGAGATCTACACGATCGGAAGAGCACACGTCTGAACTCCAGTCACnnnnnnGCACAAAAGGAAACTCACCCT) (sequences listed in supplemental table). For each sample total gDNA was amplified in multiple PCRs with an input of 10 μg per reaction. PCRs were run with the following cycling conditions: 98C for 30 s, followed by 24 cycles of 98C for 10 s and 62.6C for 75 s, and a final elongation at 65C for 5 mins. Reactions were pooled and purified with double-sided SPRI beads (Beckman Coulter, A63882). Samples were quantified on the Agilent TapeStation and pooled to generate a library with equal representation of each sample. Library was sequenced on the Illumina NextSeq 550 using a NextSeq High Output kit (75 cycles) (Illumina, 20024906). A custom sequencing primer (GTGTGTTTTGAGACTATAAGTATCCCTTGGAGAACCACCTTGTTGG) was added to the standard Illumina sequencing primer. Library runs were supplemented with PhiX DNA to increase sequence diversity. The lentiviral expression vector used for CRISPR guide expression is available on Addgene under the following name: pLGR1002 (188320). The sgRNA sequences as well as the sgRNA library cloning procedure performed is previously (PMID 38243310, PMID 27661255). sgRNA library representation was confirmed by NGS sequencing. Guide library virus was prepared using the Mega Lentivirus protocol from the Weissman Lab with modifications (https://weissman.wi.mit.edu/resources/Large_scale_lentivirus_production.pdf). 18 million LentiX 293T cells (Takara Bio, 632180) were plated on a 15 cm plate. The next day, transfection mixture was prepared using 125 μl Mirus LT1 transfection reagent (Mirus Bio, MIR2304), 1.5 ml DMEM (Gibco, 11965-092), 18 μg psPax2 (packaging vector), 4 μg MD2G (env expression plasmid, VSVG), and 18 μg of lentiviral plasmid (sgRNA library). Transfection mixture was incubated for 15-20 mins at room temperature. Transfection mix was added to the 293T cells and incubated for 6 hours. After 6 hours the media was replaced with fresh 293T media supplemented with 2 μg/mL ViralBoost (ALSTEM, VB100). Lentiviral supernatant was collected 48 hours after transfection and filtered (0.45um). Lentivirus was stored at -80C until use. Lentivirus titering was performed in K562 cells prior to the screen. Multiplicity of infection (MOI) was determined by percent infection shown by BFP signal via flow cytometry. 2e5 K562 sgNT and K562 sgNPC1 cells were transduced with guide library lentivirus in a range of 5-250 μL in K562 media supplemented with 1μl/mL Polybrene (Millipore Sigma, TR-1003-G). Cells were left in virus for 24 hours and then changed to regular media. 48 hours post infection cells were analyzed on the Attune NXT flow cytometer to determine BFP fluorescence and MOI. Lentivirus for individual guide RNA experiments was produced in 6 well plates with adjusted protocol. 0.8e6 Lenti-X 293T cells per well were seeded the day before transfection. Transfection mix consists of 7.5 μL Mirus transfection reagent (MIR 2700), 100 μL DMEM (Gibco, 11965-092), 1.2 μg psPAX2, 0.3 μg MD2G, and 1.2 μg guide plasmid. Protocol follows as above.