GSM7502704: Input; Homo sapiens; ChIP-Seq
Cells were fixed with 1% formaldeyde for 15 min at room temperature and then quenched with 5 ml of PBS/GLYCINE for 5 min at room temperature. Matrigel was melted and Cells were lysed in 0.1 ml ChIP lysis buffer (50 mM Hepes-KOH, pH 7.5, 140 mM NaCl, 1% Triton X-100, and 0.1% deoxycholate with 0.1 mM PMSF and the EDTA-free Protease Inhibitor Cocktail). Samples were incubated on ice for 10 min and then centrifuged at 3,000 rpm for 3 min at 4°C. The pellet was resuspended in 500 µl lysis buffer 2 (10 mM Tris, pH 8.0, 200 mM NaCl with 0.1 mM PMSF and the EDTA-free Protease Inhibitor Cocktail) and incubated at room temperature for 10 min. Samples were centrifuged at 3,000 rpm for 5 min at 4°C. Next, the pellet was resuspended in 300 µl lysis buffer 3 (10 mM Tris, pH 8.0, 100 mM NaCl, 0.5% deoxycholate, and 0.1% SDS with 0.1 mM PMSF and the EDTA-free Protease Inhibitor Cocktail). Cells were sonicated using a Biorupter (Diagenode) for 15 min (30 s on, 1 min off). Next, 30 µl of 10% Triton X-100 was added to each tube, and then samples were centrifuged at max speed for 15 min at 4°C. The supernatant was transferred to new tubes, and the DNA concentration was quantified. Samples were precleared for 1 h at 4°C on a rotator using 8 µl protein A Dynabeads (Zymo) in ChIP lysis buffer. Samples were centrifuged at max speed for 15 min at 4°C, after which the supernatant was transferred to a new tube. 1/100 input was collected. 3 µg Antibodies were added O/N. 8 µl of protein A Dynabeads were added and chromatin was immunoprecipitated for 2h at 4°C. The following washes were performed for 15 min each by rotating for 15 min at 4°C: ChIP lysis buffer (twice), ChIP lysis buffer + 0.65 M NaCl, wash buffer (10 mM Tris-HCl, pH 8.0, 250 mM LiCl, 0.5% NP-30, 0.5% DOC, and 1 mM EDTA, pH 8.0), and TE (10 mM Tris-HCl, pH 8.0, and 1 mM EDTA, pH 8.0). Beads were incubated with RNASEA solution (100ul TE/2ul Rnasi A (10mg/ml). DNA was eluted by adding to the beads 100ul of TES (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, pH 8.0, and 1% SDS) for 15 min at 65°C with brief vortexing every 2 min. Reversal of cross-linking was performed by incubating samples overnight at 65°C: transfer into 0.5ml tubes and place in termocycler. Proteins were digested using 150ug of proteinase K (0.5ul proteinase K) by incubating at 37°C for 5h. Finally, the DNA was purified using the Zymo Research Kit and eluted 2 times in 40ul H20. Sonicated DNA underwent End repair and A-tailing steps (MGI Tech Co., Shenzen, China). Adapters ligation was performed following the instructions of the MGIEasy DNA Adapters kit, and cleaned up with the provided DNA Clean Beads. PCR amplification was carried out and cleaned-up again using magnetic beads. After quality control of DNA, purified PCR products were denaturated and ligated to generate single-strand circular DNA libraries. Barcoded libraries were then pooled in equal amounts to make DNA Nanoballs (DNB) DNA nanoballs