GSM8229662: E_Foxf2-Flag_ChIP_r1; Homo sapiens; ChIP-Seq
ChIPseq was done essentially as previously described (Cernilogar et al. Nucleic Acids Res, 2019  PMID 31350899). Briefly, two million cross-linked cells (1% formaldehyde, 10min RT) were lysed in 100 μl Buffer-B-0.3 (50mM Tris-HCl, pH 8.0, 10 mM EDTA, 0.3% SDS, 1x protease inhibitors, Roche) and sonicated in a microtube (Covaris, Cat# 520045) using a Covaris S220 device until most of the DNA fragments were 200-500 base pairs long (settings: temperature 4°C, duty cycle 2%, peak incident power 105 Watts, cycles per burst 200). After shearing, lysates were centrifuged (10min, 4°C, 12000g) and supernatant diluted with 1 volume of Dilution Buffer (1mM EGTA 300 mM NaCl, 2% Triton x-100, 0.2% sodium deoxycholate, 1x protease inhibitors, Roche). Sonicated chromatin was then incubated for 4 h at 4°C on a rotating wheel with 3 μg of anti-Flag antibody (Sigma Cat# F1804) conjugated to 30 µl of Protein-G Dynabeads (Thermofisher). Beads were washed five times with Buffer-A (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.5 mM EGTA,1% Triton X-100, 0.1% SDS, 0.1% Na-deoxycholate, 140 mM NaCl, 1x protease inhibitors) and once with Buffer-C (10 mM Tris-HCl, pH 8.0, 10 mM EDTA). Beads were then incubated with 70 μl elution buffer (0.5% SDS, 300 mM NaCl, 5 mM EDTA, 10 mM Tris HCl pH 8.0) containing 2 μl of Proteinase K (20mg/ml) for 1 h at 55°C and 8 h at 65°C to revert formaldehyde crosslinking, and supernatant was transferred to a new tube. Another 30 μl of elution buffer was added to the beads for 1 minute and eluates were combined and incubated with another 1 μl of Proteinase K for 1 h at 55°C. Finally, DNA was purified with SPRI AMPure XP beads (Beckman Coulter) (sample-to-beads ratio 1:2). Purified DNA was used as input for library preparation with Thruplex DNA-seq Kit (Takara, Cat# R400674) and processed according to the manufacturer´s instruction.