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| Status |
Public on Aug 22, 2019 |
| Title |
MeCP2_ChIPRx_HCT116_rep2 |
| Sample type |
SRA |
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| Source name |
colon cancer cell line
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| Organism |
Homo sapiens |
| Characteristics |
tissue: colon cancer cell line
cell line: HCT116
spike-in reference: Drosophila melanogaster
spikein_mix_ratio: 3 to1
chip antibody: MeCP2 (Diagenode, pAb-052-050)
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| Treatment protocol |
N/A
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| 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.
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| 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 4C) 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.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
HCT116
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| 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.
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| Submission date |
Aug 21, 2019 |
| Last update date |
Aug 22, 2019 |
| Contact name |
Wooje Lee |
| E-mail(s) |
ntinamu001@gmail.com
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| Organization name |
Chosun University
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| Department |
Cellular and Molecular Medicine
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| Street address |
309, Pilmun-daero, Dong-gu
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| City |
Gwangju |
| ZIP/Postal code |
61452 |
| Country |
South Korea |
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| 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] |
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| Relations |
| BioSample |
SAMN12616426 |
| SRA |
SRX6748000 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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