title: GSM5252654: H9 hTSC TEAD1 CUT&Tag rep 1; Homo sapiens; OTHER
library_construction_protocol: CUT&Tag: CUT&Tag was performed as previously described. Briefly, 200,000 cells per sample were washed in Wash Buffer [1 mL 1 M HEPES pH 7.5 (Sigma Aldrich, H3375), 1.5 mL 5 M NaCl (Sigma Aldrich, S5150), 12.5 μL 2 M Spermidine (Sigma Aldrich, S2501), 1 Roche Complete Protease Inhibitor EDTA-Free tablet (Sigma Aldrich, 5056489001), and bring the final volume to 50 mL with dH2O], captured with Concanavalin A beads (Bangs Laboratories, BP531), and incubated with primary antibody at 4°C overnight. The cells were then incubated with secondary antibody at room temperature for an hour. The following antibodies were used: TEAD1 (Cell Signaling Technology, 12292S); guinea pig-anti-rabbit (Antibodies online, ABIN101961). After washing off the unbound antibodies with Dig-Wash Buffer [mix 400 μL 5% Digitonin (EMD Millipore, 300410) with 40 mL Wash Buffer], the pA-Tn5 adapter complex (kindly provided by Dr. Steve Henikoff) were added at 1:200 and incubated at room temperature for an hour. The cells were washed again with Dig-300 Buffer [1 mL 1 M HEPES pH 7.5, 3 mL 5 M NaCl, 12.5 μL 2 M Spermidine, 100 μL 5% Digitonin, 1 Roche Complete Protease In-hibitor EDTA-Free tablet, and bring the final volume to 50 mL with dH2O], and incubat-ed in 300 μL Tagmentation Buffer [5 mL Dig-300 Buffer and 50 μL 1 M MgCl2 (Sigma Aldrich, M8266)] per sample at 37°C for an hour. The reaction was stopped and the DNA solubilized by adding 10 μL 0.5 M EDTA (Research Organics, 3002E), 3 μL 10% SDS (Sigma Aldrich, L4509), and 2.5 μL Proteinase K (Thermo Fisher, EO0492) per sample, and incubated at 50°C for an hour. The DNA was then extracted, excess RNA digested with RNase A (Thermo Fisher, EN0531), and PCR amplified with i5 and i7 in-dexing primers. The PCR product was cleaned up with AMPure XP beads, and the size distribution and concentration were confirmed using Tapestation.
attributes.tag: GEO Accession
attributes.value: GSM5252654