title: GSM5542579: 786-O_H3K4me3_ChIP_input_Rep1; Homo sapiens; ChIP-Seq
library_construction_protocol: ChIP assay was performed on 786-O cells with anti-JMJD6 (Abcam, ab64575) and anti-H3K4me3 (Abcam, ab8580) antibody. Briefly, samples were cross-linked with 1% formaldehyde on a shaker for 10 min, followed by adding 125 mM glycine to quench unreacted formaldehyde. Samples were collected by centrifuging at 1500 × g, 4°C for 10 min (50 ml tube, 20 150 mm dishes per sample), followed by lysis and sonication (in 5 ml lysis buffer with 1 ml 0.5 mm glass beads; 30 s on 30 s off/cycle, 50 cycles in total). Meanwhile, incubate dynabeads with JMJD6 or H3K4me3 antibody at 4°C (100 ml beads using 10 mg antibody), rotating overnight. The sonicated samples were then incubated with antibody-bound dynabeads overnight at 4°C. Wash beads 8 times by applying magnetic stand, spin to collect samples, and elute 30 min at 65°C, further reverse crosslink at 65°C overnight. Digest RNA using RNase (Roche, 11119915001) at 37°C for 1 h, and digest protein using Proteinase K (Sigma-Aldrich, 3115828001) at 55°C for 2 h. Purify DNA using Qiagen PCR purification kit. We prepared DNA library using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (New England Biolabs, E7645S) combined with NEBNext Multiplex Oligos for Illumina (New England Biolabs, E7335S) according to manufacturer's instructions. The quality of prepared DNA library was checked by applying high sensitivity D1000 reagents and corresponding screen tape (Agilent Technologies, 5067-5585/5584), followed by sequencing at the CRI's Sequencing Facility at UT Southwestern.