title: GSM4041344: Input_ChIPRx_DKO1_rep1; Homo sapiens; ChIP-Seq
library_construction_protocol: HCT116 or DKO1 cells (density of 0.5~0.6 x 106 cells/ml) were washed with 10 ml of PBS and cross-linked with 1% formaldehyde for 5 min. The crosslinking reactions were quenched with 10x Glycine stop solution. The cross-linked HCT116 or DKO1 cells were lysed in lysis buffer (5 mM PIPES, 85 mM KCl, 0.5% IGEPAL CA-630, pH 8.0) using a dounce homogenizer (Kimble-Kontes. 885300-0002) to aid in nuclei release. The released nuclei was pelleted by centrifugation (10 min, 5000 rpm at 4C) and stored at -80°C. As a reference exogenous genome, Drosophila S2 cells were cross-linked with 1% formaldehyde for 5 min and were quenched with 10x Glycine stop solution. The Drosophila S2 cells were then washed 3 times with ice cold PBS. Washed cell pellets were flash frozen and stored at -80°C. For each ChIP-Rx experiment, HCT116 or DKO1 cells and drosophila S2 cells were combined with ratio of 3:1 and sonicated for 15 min with 30 sec intervals to shear genomic DNA using Bioruptor XL (Diagenode). The sheared genomic DNA was evaluated by electrophoresis for their size ranging between 200 bp and 300 bp. Each sheared genomic DNA preparation was incubated with H3K27me3 (Diagenode, pAb-069-050) or MeCP2 (Diagenode, pAb-052-050). After pull down, genomic DNA in the complex was reverse cross-linked by DNase-free Proteinase K and purified. For ChIP-seq experiments, H3K27me3 or MeCP2 ChIPed, or input DNA were used to prepare ChIP-seq libraries as described (NEXTflex ChIP-seq kit, Bio Scientific, NOVA 5143-01 and NEXTflex ChIP-seq Barcodes-6, NOVA 514120) followed by high throughput sequencing with Illumina NextSeq500.