GSM8657776: BC776_H3K27me3; Homo sapiens; ChIP-Seq
Cells were crosslinked with 1% formaldehyde and the reaction was quenched with glycine. Dynabeads protein G (Thermo Fisher Scientific) were washed with cold IP-100 buffer (25 mM Tris-HCl pH 7.5, 100 mM KCl, 5 mM MgCl2, 10% v/v glycerol, 0.1% NP-40 alternative [Calbiochem, 492016-100ML], 200 µM PMSF) and incubated with antibody for 6-8h at 4°C while rotating. Cells were then sonicated in sonication buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% v/v Triton X-100, 0.1% w/v sodium deoxycholate, 1x EDTA-free proteinase inhibitor cocktail [PIC] [Roche], 1% v/v SDS) to a fragment size of approximately 500 bp. Sonication buffer without SDS was added to dilute SDS to 0.1% v/v. Antibody-coated beads were added to the rest of the sample, and IP was carried out O/N at 4°C while rotating. Beads were washed as follows: Twice with sonication buffer without SDS, twice with wash buffer A (50mM HEPES-KOH pH 7.5, 500 mM NaCl, 1 mM EDTA, 1% v/v Triton X-100, 0.1% w/v sodium deoxycholate, 0.1% v/v SDS, 1x PIC), twice with wash buffer B (20 mM Tris-HCl pH 8.1, 1 mM EDTA, 250 mM LiCl, 0.5% v/v IGEPAL CA-630, 0.5% w/v sodium deoxycholate, 1x PIC) and twice with TE buffer (10 mM Tris-HCl pH 8.1, 1 mM EDTA), followed by addition of elution buffer (50 mM Tris-HCl pH 8.1, 1 mM EDTA, 1% v/v SDS). Samples were vortexed and incubated at 65°C, after which the first eluate was taken. The elution process was then repeated. NaCl and RNase A were added, and samples were incubated O/N at 65°C. EDTA and proteinase K were added, and samples were incubated at 42°C for 2h. Extraction with 25:24:1 phenol:chloroform:isoamyl alcohol (Sigma, P2069-400ML) was performed twice. Glycogen, sodium acetate pH 5.6 and two volumes of 100% ice-cold ethanol were added, after which DNA was precipitated at -20°C. DNA was washed and then resuspended in 10 mM Tris-HCl pH 7.5. Cells were crosslinked with 1% formaldehyde and the reaction was quenched with glycine. Dynabeads protein G (Thermo Fisher Scientific) were washed with cold IP-100 buffer (25 mM Tris-HCl pH 7.5, 100 mM KCl, 5 mM MgCl2, 10% v/v glycerol, 0.1% NP-40 alternative [Calbiochem, 492016-100ML], 200 µM PMSF) and incubated with antibody for 6-8h at 4°C while rotating. Cells were then sonicated in sonication buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% v/v Triton X-100, 0.1% w/v sodium deoxycholate, 1x EDTA-free proteinase inhibitor cocktail [PIC] [Roche], 1% v/v SDS) to a fragment size of approximately 500 bp. Sonication buffer without SDS was added to dilute SDS to 0.1% v/v. Antibody-coated beads were added to the rest of the sample, and IP was carried out O/N at 4°C while rotating. Beads were washed as follows: Twice with sonication buffer without SDS, twice with wash buffer A (50mM HEPES-KOH pH 7.5, 500 mM NaCl, 1 mM EDTA, 1% v/v Triton X-100, 0.1% w/v sodium deoxycholate, 0.1% v/v SDS, 1x PIC), twice with wash buffer B (20 mM Tris-HCl pH 8.1, 1 mM EDTA, 250 mM LiCl, 0.5% v/v IGEPAL CA-630, 0.5% w/v sodium deoxycholate, 1x PIC) and twice with TE buffer (10 mM Tris-HCl pH 8.1, 1 mM EDTA), followed by addition of elution buffer (50 mM Tris-HCl pH 8.1, 1 mM EDTA, 1% v/v SDS). Samples were vortexed and incubated at 65°C, after which the first eluate was taken. The elution process was then repeated. NaCl and RNase A were added, and samples were incubated O/N at 65°C. EDTA and proteinase K were added, and samples were incubated at 42°C for 2h. Extraction with 25:24:1 phenol:chloroform:isoamyl alcohol (Sigma, P2069-400ML) was performed twice. Glycogen, sodium acetate pH 5.6 and two volumes of 100% ice-cold ethanol were added, after which DNA was precipitated at -20°C. DNA was washed and then resuspended in 10 mM Tris-HCl pH 7.5.