title: GSM6925320: K562-ReChIP-with-clear-H3K4me3; Homo sapiens; ChIP-Seq
library_construction_protocol: Cells were cross-linked with 1% formaldehyde for 15 min at room temperature, and cross-linking was stopped by the addition of 0.125 M glycine. Cells were lysed in 1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.0, and protease inhibitors. Lysates were sonicated in a Covaris ultrasonicator to achieve a mean DNA fragment size of 500 bp. Samples were diluted 1:10 in modified  RIPA buffer (1% Triton X-100, 0.1% deoxycholate, 90 mM NaCl, 10 mM Tris-HCl, pH 8.0, and protease inhibitors) then pre-cleared with Protein A Dynabeads (Life Technologies). Chromatin was then incubated with pre-bound anti-H3K27me3-bead mixture rotating overnight at 4 °C. After washing, samples were eluted from beads in Elution Buffer 1 (50 mM Tris pH 8.0, 1% SDS, 10 mM DTT).  Eluates were diluted 1:20 in modified RIPA buffer , 10% was kept back as primary ChIP, and then pre-cleared with Protein A Dynabeads rotating overnight at 4 °C. Samples were washed and incubated with second pre-bound anti-H3K4me3-bead mixture and rotated again overnight at 4°C. Samples were washed and eluted in Elution Buffer 2 (1% SDS, 0.1 M NaHCO3).  Samples were reverse cross-linked overnight and purified with MinElute PCR purification kit (Qiagen). Sequencing libraries were prepared from eluted DNA using Rubicon ThruPLEX DNA-seq kit.