GSM8140620: Donor B, NTC CRISPRi; Homo sapiens; OTHER
Cell pellets were thawed, fixed with 1% PFA, and re-pelleted. Cell pellets were resuspended with 500ul 4C lysis buffer (50mM Tris-HCl pH 7.5, 150mM NaCl, 5mM EDTA, 0.5% NP-40 (IGEPAL CA-630), 1% Triton-X100, and 1X protease inhibitors (ThermoFisher #1862209)). Pellets were pipetted vigorously and lysed on ice for 10 minutes. Pellets were centrifuged 750g, 5 minutes, 4°C and washed twice with cold PBS. Nuclear pellets were resuspended in H2O and 1X rCutSmart buffer (NEB #R3104T). 0.25% SDS and 2.5% Triton-X100 were added for denaturation at 37°C for one hour on a thermomixer set to 900 rpm. Genomic DNA was digested with 400UI-600UI HindIII-HF (NEB #R3104T) overnight before heat inactivation. Digested genomic DNA was ligated by T4 DNA ligase system (NEB #M0202T) at 25°C 4 hours. The mixture was digested by proteinase K (ThermoFisher #EO0491) and RNase (Roche #11119915001) and purified by phenol-chloroform. DNA pellets were resuspended in TE buffer and subjected to DpnII secondary digestion overnight (200UI, NEB #R0543T) before heat inactivation. DNA was again ligated using the T4 DNA ligase system (NEB #M0202T) at 25°C 4 hours and then pelleted with 60mM sodium acetate, 3ug/ml glycogen, and 70% Ethanol. Two probe sets spanning the entire Stim-Responsive CiRE were tested, but only the probe covering the latter half of the enhancer region (which aligns with maximum CRISPRi responsiveness) yielded sufficiently diverse libraries and is included here. PCR was performed on 200ng DNA with CM_oligo_8 and CM_oligo_9 using Platinum™ SuperFi™ DNA Polymerase system (ThermoFisher #12351010) with the program [98°C 10”, 52°C 10”, 72°C 1'] x 30 cycles. Final amplified libraries were purified with SPRI clean-up, quantified, and sequenced.