title: GSM5230330: CRISPR_Donor3_CTLA4_high; Homo sapiens; OTHER
library_construction_protocol: After sorting, cells were washed with PBS, counted, pelleted, and resuspending at up to 5E6 cells per 400 µl of lysis buffer (1% SDS, 50 mM Tris, pH 8, 10 mM EDTA). The remaining protocol reflects additives/procedures performed per each 400 µl of sample. 16µl of NaCl (5M) was added, and the sample was incubated on a heat block overnight at 66°C. The next morning, 8µl of RNAse A (10mg/ml, resuspended in ddH­2O) (Zymo, Cat #E1008) was added, and the sample was vortexed briefly, and incubated at 37°C for 1 hour. Next, 8µl of Proteinase K (20mg/ml) (Zymo, Cat #D3001) was added, the sample was vortexed briefly, and incubated at 55°C for 1 hour. A phase lock tube (Quantabio, Cat #2302820) was prepared for each sample by spinning down the gel to the bottom of the tube at 20,000g for 1 minute and then 400µl of Phenol:Chloroform:Isoamyl Alcohol (25:24:1) was added to each tube. 400µl of the sample was then added to the phase lock tube and the tube was shaken vigorously. The sample was centrifuged at maximum speed at room temperature for 5 minutes. The aqueous phase was transferred to a low-binding eppendorf tube (Eppendorf, Cat #022431021) and then 40µl of Sodium Acetate (3M), 1µl GlycoBlue (Invitrogen, Cat # AM9515), and 600µl of room temperature isopropanol was added. The sample was then vortexed and stored at -80°C for 30 minutes or until the sample had frozen solid. Next the sample was centrifuged at maximum speed at 4°C for 30 minutes, the pellet was washed with fresh 70% room temperature Ethanol, and allowed to air dry for 15 minutes. Pellets were then resuspended in Zymo DNA elution buffer (Zymo, Cat No: D3004-4-10), and placed on the heat block at 65°C for 1 hour to completely dissolve the genomic DNA. sgRNA was amplified and barcoded from the genomic DNA according to the protocol by Joung et al. (Joung et al. 2017). Up to 2.5 µg of genomic DNA were added to each 50 µL reaction, which included 25 µL of NEBNext Ultra II Q5 master mix (NEB, Cat #M0544L), 1.25 µL of the 10 µM forward primer and 1.25 µL of the 10 µM reverse primer, and H2O to 50 uL. The following PCR cycling conditions were used: 98°C for 3 minutes, followed by 23 cycles at 98°C for 10 seconds, 63°C for 10 seconds, and 72°C for 25 seconds, and ending with 2 minutes at 72°C. Samples were then cleaned and concentrated in Zymo Spin-V columns (Zymo, Cat #C1016-50) following Joung et al., and eluted in 150 uL of Zymo DNA elution buffer. Up to 2 µg of each library were loaded on a 2% agarose gel, and the band at ~250 base pairs was extracted using the Zymoclean Gel DNA recovery kit (Zymo, Cat #D4008). The concentration of each sample was then measured using the Qubit dsDNA high sensitivity assay kit (Thermo Fisher Scientific, Cat #Q32854). Samples were then sequenced on an Illumina HiSeq 4000 using 10-30% PhiX (Illumina, Cat #15017872), and a custom sequencing primer.