title: GSM8804780: H3K4me3_PRDM9_rep2; Homo sapiens; ChIP-Seq
library_construction_protocol: 3 x 10^6 cells were cross-linked in 1% methanol-free formaldehyde at room temperature for 10 min, and then quenched with 0.114 M Glycine. Fixed cells were resuspended in 300 uL High Salt Lysis/Sonication Buffer (800 mM NaCl, 25 mM Tris pH 7.5, 5 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, 1 x protease inhibitor cocktail) and frozen in -80°C. 20 uL Dynabeads Protein G (Thermo Fisher Scientific) was first incubated with 2 ug antibodies in 20 uL Chromatin Dilution Buffer (25 mM Tris pH 7.5, 5 mM EDTA, 1% Triton X-100, 0.1% SDS, 1 x protease inhibitor cocktail) at room temperature for 3 hours with rotation. Fixed cells were transferred to a Bioruptor Pico Microtube (Diagenode) and sonicated for 10 cycles (30 s on/off) in Bioruptor (Diagenode) at 4°C. 1 mL Chromatin Dilution Buffer was added to sonicated cells, and samples were spun at 13600 x g for 30 min at 4°C to pellet insoluble material. If appropriate, 2 uL SNAP-ChIP spike-ins (EpiCypher) was added to the supernatant. 65 uL soluble chromatin was set aside as input. Dynabead/antibody mix was added to the soluble chromatin and incubated at 4°C overnight with rotation. Then, beads were washed with 1 mL Wash Buffer A (140 mM NaCl, 50 mM HEPES pH 7.9, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate), Wash Buffer B (500 mM NaCl, 50 mM HEPES pH 7.9, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate), and Wash Buffer C (20 mM Tris pH 7.5, 1 mM EDTA, 250 mM LiCl, 0.5% NP-40 Alternative, 0.5% sodium deoxycholate) once each, and TE Buffer (10 mM Tris pH 7.5, 1 mM EDTA) twice. Bound DNA was eluted twice with 100 uL Elution Buffer (10 mM Tris pH 7.5, 1 mM EDTA, 1% SDS) at 65°C for 5 min. Eluted DNA and input samples were incubated with 4 ug RNase A (Millipore Sigma) at 65°C overnight to reverse cross-linking and digest any contaminating RNA. ChIP and input samples were further digested with 40 ug Proteinase K (NEB) at 45°C for 2 hours and then purified with the Zymo ChIP DNA Clean & Concentrator Kit (Zymo Research) following the manufacturer's instructions. ChIP-seq libraries were prepared using either the KAPA HyperPrep Kit (Roche, KK8504) or the Watchmaker DNA Library Prep Kit (#7K0103-096).
identifiers.namespace: GEO
identifiers.id: GSM8804780_r1