GSM8112105: SUM149, ROSA26KO, DMSO, KDM5B_IP, rep2; Homo sapiens; ChIP-Seq
Cells were directly fixed in 15 cm cell culture plates. For ChIP-seq of histone modifications (H3K4me3), samples were crosslinked in fixing buffer (50 mM HEPES-NaOH pH 7.5, 100 mM NaCl, 1 mM EDTA) containing 1% PFA (Electron Microscopy Sciences, 15714) for 10 minutes at room temperature (i.e., single fixation). For ChIP-seq of chromatin binding proteins (KDM5A, KDM5B, ZBTB7A), samples were first crosslinked in PBS containing 2 mM DSG (Fisher Scientific 20593) for 30 minutes at room temperature. DSG was then removed, and the samples were further crosslinked with fixing buffer (50 mM HEPES-NaOH pH 7.5, 100 mM NaCl, 1 mM EDTA) containing 1% PFA for 10 minutes at 37 oC (i.e., double fixation). After PFA fixation, all samples were quenched with glycine at a final concentration of 0.125 M for 5 minutes at room temperature. Cells were washed and harvested in ice cold PBS. Nuclei were extracted by first resuspending the cells in lysis buffer for 10 minutes at 4C (50 mM HEPES-NaOH pH 8, 140 mM NaCl, 1 mM EDTA, 10% Glycerol, 0.5% IGEPAL CA-630, 0.25% Triton X-100), 1 mL of lysis buffer per 5 million cells. Nuclei were pelleted and washed once in wash buffer (10 mM Tris-HCl pH 8, 200 mM NaCl, 1 mM EDTA), 1 mL of wash buffer per 10 million (histone modification ChIP) or 25 million cells (chromatin modification ChIP). The nuclei were then pelleted and resuspended in 1 mL of shearing buffer (10 mM Tris-HCl pH 8, 1 mM EDTA, 0.1% SDS), containing 10 million (histone modification ChIP) or 25 million cells (chromatin modification ChIP). Samples were transferred to 1 mL AFA Fiber tubes (Covaris 520130), and sonicated in a Covaris E220 sonicator with the following settings: Peak Incident Power 150, Duty Cycles 5%, cycles per Burst 200. Histone modification ChIP samples were sonicated for 900 seconds. Chromatin binding protein ChIP samples were sonicated for 1,200 seconds. After sonication, Triton X-100 and NaCl were added to a final concentration of 1% Triton X-100 and 150 mM NaCl. The samples were then pre-cleared with 50 µL of Dynabeads Protein G (Fisher Scientific, 10004D) for 1 hour at 4 oC. For histone modification ChIP, primary antibodies were added to 1 mL of pre-cleared chromatin from ~10 million cells (H3K4me3 = Diagenode C154100003 at 5 µg/mL) and incubated at 4 oC overnight. For chromatin binding protein ChIP, pre-cleared samples were aliquoted and volumes adjusted using shearing buffer containing 1% Triton X-100 and 150 mM NaCl so that 1 mL contained pre-cleared chromatin from ~10 million cells. Primary antibodies were added to each aliquot and incubated  at 4 oC overnight (ZBTB7A = SantaCruz sc-33683X at 5 µg/mL, KDM5A = Abcam ab70892 at 4 µg/mL, KDM5B = Novus/sdix 22260002 at 5 µg/mL). After overnight immunoprecipitation, crosslinked complexes were precipitated with  Dynabeads G (Fisher Scientific, 10004D) for 2 hours at 4 oC. The beads were then washed once with low salt wash buffer (20 mM Tris-HCl pH 8, 150 mM NaCl, 0.1% SDS, 1% Triton-X 100, 2 mM EDTA) for 5 minutes at 4 oC, once with high salt wash buffer (20 mM Tris-HCl pH 8, 500 mM Nacl, 0.1% SDS, 1% Triton-X 100, 2 mM EDTA) for 5 minutes at 4 oC, once with wash buffer (10 mM Tris-HCl pH 8, 250 mM LiCl, 1% IGEPAL CA-630, 1% Sodium Deoxycholate, 1 mM EDTA) for 5 minutes at 4 oC, and once with TE buffer (10 mM Tris-HCl pH8, 1 mM EDTA) quickly at room temperature. DNA was eluted from the beads in 1% SDS, 100 mM NaHCO3 for 30 minutes at room temperature. Crosslinks were reversed at 65 oC overnight. RNA and protein were digested with 0.2 mg/mL RNase A for 30 minutes at 37 oC followed by 0.2 mg/mL Proteinase K for 1 hour at 55 oC. DNA was extracted with 300 µl of phenol/chloroform at pH8 and centrifuged at 20,000g for 5 min at RT. DNA in the upper layer was then precipitated with 300 µL isopropanol, 100 µL of 2 M sodium perchlorate, and 5 µl glycogen and centrifuged at 20,000g for 10 min at RT. Pelleted DNA was washed twice with 70% ethanol and resuspended in 20 µl low TE. Libraries were prepared with the automated Swift 2S ligation chemistry and sequenced to 40M 50 bp reads pairs.