GSM9648644: Donor2, low TNF expression bin; Homo sapiens; OTHER
Sorted cells were pelleted and resuspended at up to 5x10^6 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 reaction. 16 µl of NaCl (5M; Fisher Scientific; AM9760G) was added, and the sample was incubated on a heat block overnight at 66°C. The next morning, 8 µl of RNAse A (10mg/ml; Thermo Fisher; FEREN0531) was added, and the sample was vortexed briefly, and incubated at 37°C for 1 hour. Next, after 8 µl of Proteinase K (20mg/ml; ThermoFisher; AM2548) was added, the sample was vortexed briefly and incubated at 55°C for 1 hour. Homemade phase lock tubes were prepared for each sample by spinning 200 µl sterilized high vacuum grease gel (DuPont; B0857KKVR8) to the bottom of a 1.5 ml tube at 20,000 g for 1 minute and then 400 µl of Phenol:Chloroform:Isoamyl Alcohol (25:24:1; Fisher Scientific; 15-593-031) was added to each tube. 400 µl 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 40 µl of Sodium Acetate (3M; VWR; 46-033-CI), 1µl GlycoBlue (Invitrogen; AM9515), and 600µl of room temperature isopropanol (Fisher Scientific; 32727-0010) 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; D3004-4-10), and placed on the heat block at 65°C for 1 hour to completely dissolve the genomic DNA. The sgRNA cassette was amplified and barcoded from the genomic DNA as initially described by Joung et al82. 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; 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. Due to differences in sgRNA scaffold in the respective plasmid backbone, different reverse primer sets were used for CD80 and TNF screens. 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. Amplicons were purified using SPRIselect beads following manufacturer's protocols (Beckman). The concentration of each sample was then measured using the Qubit dsDNA high sensitivity assay kit (Thermo Fisher Scientific, Cat #Q32854).