title: GSM4817491: K562_CRISPR_IGF1KO_H3K27me3_rep2_ChIPSeq; Homo sapiens; ChIP-Seq
library_construction_protocol: 5 million cells per ChIP was harvested, washed twice and resuspended in PBS. The cells were crosslinked with 1% methanol-free formaldehyde (Thermo Scientific) at room temperature for 10 minutes, followed by quenching with glycine for 5 minutes at room temperature. The cell pellet was washed with twice with PBS supplemented with 1mM PMSF (Sigma). The fixed cell pellet was lysed in 1% SDS lysis buffer (50 mM HEPES pH7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Sodium Deoxycholate, 1% SDS) supplemented with protease inhibitor cocktail tablet (Roche), and sonicated using Bioruptor (Diagenode) and 200-500bp DNA fragments were achieved. The cell lysate was cleared through centrifugation at 13,200rpm for 35 minutes at 4°C. 9 volumes of dilution buffer (50 mM HEPES pH7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Sodium Deoxycholate) was added to the lysate to achieve 0.1% SDS lysis buffer, and the precleared through incubation with Protein G Dynabeads (Invitrogen) overnight at 4°C, with rotation. Antibody-conjugated beads were prepared by incubating 3.5ug antibody in 15uL Protein G Dynabeads in 50uL wash buffer (PBS, 0.1% Triton X-100) overnight at 4°C, with rotation. The precleared lysate was added into the prepared antibody-conjugated beads, and was incubated overnight at 4°C, with rotation. The beads were washed thrice in 0.1% SDS lysis buffer (50 mM HEPES pH7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Sodium Deoxycholate, 0.1% SDS), once in high salt wash buffer (50 mM HEPES pH 7.5, 350 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Sodium Deoxycholate, 0.1% SDS), once in lithium chloride wash buffer (10 mM Tris pH 8.0, 250 mM LiCl, 1 mM EDTA, 0.5% Igepal- CA-630, 0.5% Sodium Deoxycholate), and once in TE buffer. The beads were then eluted in elution buffer (50 mM Tris pH 8.0, 10 mM EDTA, 1% SDS), treated with RNase A (Qiagen) with 55°C for 30 minutes, and decrosslinked with Proteinase K (Ambion) at 37°C overnight. ChIP DNA was cleaned up with QIAquick PCR purification kit (Qiagen), and quantitated using Qubit High Sensitivity dsDNA Assay (Invitrogen). 1ng of ChIP DNA was used for the construction of DNA library for Illumina HiSeq 4000 NGS sequencing using ThruPLEX DNA-seq 48D Kit (Rubicon) as per protocol. Final libraries were quantitated and pooled at the same molarity using Agilent High Sensitivity DNA Kit (Agilent).