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Sample GSM4041353 Query DataSets for GSM4041353
Status Public on Aug 22, 2019
Title MeCP2_ChIPRx_DKO1_rep2
Sample type SRA
 
Source name colon cancer cell line
Organism Homo sapiens
Characteristics tissue: colon cancer cell line
cell line: DKO1
spike-in reference: Drosophila melanogaster
spikein_mix_ratio: 3 to1
chip antibody: MeCP2 (Diagenode, pAb-052-050)
Treatment protocol N/A
Growth protocol HCT116 cell line and DNMT1 (Δexons3-5/Δexons3-5); DNMT3B (-/-) of HCT116 (horizon, HD R02-079), named as DKO1, were maintained in RPMI 1640 (Sigma, R8758), supplemented with 10% FBS (Welgene, S001-01) and Penicillin/Streptomycin (Lonza, 17-602E) at 5% CO2 in 37°C. Drosophila S2 cells (ATCC, CRL-1963) were cultured in Schneider’s Drosophila Medium(Gibco, 21720024) supplemented with 10% fetal bovine serum (Welgene, S001-01) in 25°C.
Extracted molecule genomic DNA
Extraction 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.
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina NextSeq 500
 
Description DNMT1 (Δexons3-5/Δexons3-5); DNMT3B (-/-) of HCT116
Data processing The sequences were separately aligned to either human genome(hg19) using Bowtie 2 with the options --sensitive --score-min L,-1.5,-0.3. PCR duplicate are removed for further analysis.
PCR duplicate are removed using samtools rmdup for further analysis.
Genome_build: hg19
Supplementary_files_format_and_content: Input normalized bigwig files were generated using bamCompare of deepTools package with the options –normalizeUsing CPM –extendReads 200.
 
Submission date Aug 21, 2019
Last update date Aug 22, 2019
Contact name Wooje Lee
E-mail(s) ntinamu001@gmail.com
Organization name Chosun University
Department Cellular and Molecular Medicine
Street address 309, Pilmun-daero, Dong-gu
City Gwangju
ZIP/Postal code 61452
Country South Korea
 
Platform ID GPL18573
Series (2)
GSE122366 MeCP2 regulates genome-wide gene expression through recognition of H3K27me3
GSE136118 MeCP2 regulates gene expression through direct interaction with H3K27me3 [ChIP_human: HCT116_vs_DKO1]
Relations
BioSample SAMN12616424
SRA SRX6748002

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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