title: GSM3465069: H3K27me3_ChIPRx_GSK343_rep1; Homo sapiens; ChIP-Seq
library_construction_protocol: The SH-SY5Y cells were washed with 10ml of PBS and cross-linked with 1% formaldehyde for 5minutes.   The crosslinking reactions were quenched with 10x Glycine stop solution. The cross-linked SH-SY5Y 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 (10min, 5000rpm at 4°C). As a reference exogenous genome, Drosophila S2 cells were cross linked with 1% formaldehyde for 5 minutes and were quenched with 10x Glycine stop solution. The Drosophila S2 cells were then washed 3 times with ice cold PBS. For each ChIP-Rx experiment, SH-SY5Y cells and drosophila S2 cells ratio of 3:1, 12 million crosslinked SH-SY5Y nuclei and 4 million crosslinked S2 cells were combined and sonicated for 15 minutes with 30 second intervals to shear genomic DNA using Bioruptor XL (Diagenode). Each sheared genomic DNA preparation was split into two equal portions and incubated with either H3K27me3 (Diagenode, pAb-069-050), or MeCP2 (Diagenode, pAb-052-050). A magnetic bar was used to pull down antibody corresponding protein-genomic DNA complex. Genomic DNA in the complex was reverse cross-linked by DNase-free Proteinase K and purified. H3K27me3 ChIPed or input DNA were used to prepare the ChIP-Seq library as described (NEXTflex ChIP-Seq kit, Bio Scientific, NOVA 5143-01 and NEXTflex ChIP-Seq Barcodes-6, NOVA 514120) followed by high throughput sequencing.
attributes.tag: GEO Accession
attributes.value: GSM3465069