GSM3896239: EPZ1_H3K27me3; Homo sapiens; ChIP-Seq
To purify nuclei, thawed cells were washed 3x with PBS, 2x with Buffer N (15 mM Trizma Base pH 7.5, 15 mM NaCl, 60 mM KCl, 5 mM MgCl2, 1 mM CaCl2, 8.5% sucrose, 1 mM DTT, 200 uM PMSF, 50 ug/mL BSA, and protease inhibitor), and lysed with 2x Lysis Buffer (0.6% NP-40 in Buffer N). Nuclei were layered over a 30% sucrose cushion, pelleted at 1300 rcf, and resuspended in Buffer N. Crude chromatin concentration was determined by sonicating 2 uL of nuclei in 18 uL NaCl (2 M) and measuring DNA concentration with a Nanodrop; nuclei equivalent to 50 ug of DNA were aliquoted and spiked with 5 uL of a SNAP-ChIP k-MetStat panel (Epicypher). 1 U MNase (25U/uL) was added per 4.275 ug chromatin and incubated at 37°C for 12 minutes with shaking. Mnase digestion was stopped with 1/10 volume MNase Stop Buffer (10x, 0.1 M EGTA) followed by 1/8 volume NaCl (5 M) to lyse nuclei and release digested chromatin. After centrifugation, soluble chromatin was added to 33 mg of rehydrated ceramic hydroxyapatite (CHT) resin and rotated for 10 min at 4°C. The CHT Resin:Chromatin mix was added to a centrifugal filter unit and washed 4x with HAP Wash Buffer 1 (5 mM NaPO4 pH 7.2, 600 mM NaCl, 1 mM EDTA, and 200 uM PMSF), 4x with Hap Wash Buffer 2 (5 mM NaPO4 pH 7.2, 100 mM NaCl, 1 mM EDTA, and 200 uM PMSF), and eluted with three successive additions of HAP Elution Buffer (500 mM NaPO4 pH7.2, 100 mM NaCl, 1 mM EDTA, and 200 uM PMSF). DNA concentration was again measured by sonication and adjusted to 20 ug/mL with ChIP Buffer 1 (25 mM Tris pH 7.5, 5 mM MgCl2, 100 mM KCl, 10% glycerol, 0.1% NP-40, 200 uM PMSF and 50 ug/mL BSA). Antibody:bead conjugates were prepped by adding 5 or 10 uL of Cell Signaling Technology H3K27me3 antibody (CST #9733, clone C36B11, lot 14, 102 ug/mL) to 12.5 uL of Protein A Magnetic Dynabeads (Invitrogen) that had been washed with ChIP Buffer 1. Antibody:bead conjugates were incubated on a rotator for 3 hours at 4°C, washed 2x, and resuspended with ChIP Buffer 1. Purified digested chromatin (0.75 ug as measured by DNA concentration) was added to the antibody:bead conjugates while a volume equivalent to 10% was saved for input from the purified chromatin. The bead:antibody:chromatin mixture volume was brought to 100 uL with ChIP Buffer 1 and incubated on a rotator for 17 min at 4°C. Using a magnetic rack, the mixture was washed for 10 min on a rotator at 4°C 2x with ChIP Buffer 2 (25 mM Tris pH 7.5, 5 mM MgCl2, 300 mM KCl, 10% glycerol, 0.1% NP-40, 200 uM PMSF and 50 ug/mL BSA), 1x with ChIP Buffer 3 (10 mM Tris pH 7.5, 250 mM LiCl, 1 mM EDTA, 0.5% NaDeoxycholate, 0.5% NP-40, 200 uM PMSF and 50 ug/mL BSA), 1x with ChIP Buffer 1, 1x with TE Buffer (pH 8.0), and resuspended in 50 ul of ChIP Elution Buffer. This was incubated at 55°C for 5 min, and sample was eluted from beads on a magnetic rack. Finally, 2 uL NaCl (5M), 1 uL EDTA (0.5M), and 0.5 uL Proteinase K (100x) were added to ChIP and Input samples, which were incubated overnight at 55°C. DNA was recovered using KAPA Pure beads at a 1.5X ratio on a magnetic rack. After two washes with 75% EtOH, DNA was eluted in 50 uL dH20 and DNA concentration was measured with a Qubit. Libraries were prepared according to KAPA's instructions accompanying the KAPA Hyper Prep Kit (Part# KR0961 ). Briefly, DNA was end-repaired, A-Tail extended, and KAPA adapters were ligated to DNA fragments. Adapter-ligated fragments were amplified with 11 cycles on a thermocycler, and DNA was purified using KAPA Pure Beads. Prepared libraries were submitted to the VARI Genomics Core for library QC and quantification using the Agilent 2100 Bioanalyzer and KAPA Library Quantification Kit, respectively. Libraries were sequenced on an Illumina NextSeq 500 with 2 X 75 bp paired-end reads.