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| Status |
Public on Jan 11, 2023 |
| Title |
LSD1_HCT116_WT_H3K4me3_rep1 |
| Sample type |
SRA |
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| Source name |
HCT116
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HCT116
cell type: colorectal cancer cell line
genotype: wild-type
chip antibody: H3K4me3 (Abcam, ab8580)
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| Growth protocol |
Human colorectal carcinoma HCT116 cell line was cultured in McCoy’s 5A medium supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin/streptomycin (Procell, Wuhan, China, CM-0096). Human colonic epithelial cell line NCM460 was cultured in DMEM medium (Gibco, NY, USA, C11965500BT) supplemented with 10% FBS (Lonsera, S712-012S) and 100 U/mL penicillin/streptomycin (Gibco, USA, 15140122).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
5x 106 cells were cross-linked with 1% formaldehyde for 10 minutes and quenched by 125mM glycine for 5 minutes at room temperature with gentle shaking, and washed twice with PBS. Cells were then pelleted and lysed in ice-cold lysis buffer1 (50mM HEPES-KOH, pH 7.5, 140mM NaCl, 1mM EDTA, 10% Glycerol, 0.5% NP-40, 0.25% Triton X-100) and lysis buffer 2 (10mM Tris-HCl pH 8.0, 200mM NaCl, 1mM EDTA, 0.5mM EGTA) supplemented with cOmplete protease inhibitors (Roche) for 10 minutes. The cell lysate was sonicated using Covaris M220 with 5% duty factor for 10min at 4 °C to shear DNA to fragments (200~1000 bp). Soluble chromatin was diluted in shearing buffer (1mM EDTA, 10mM Tris-HCl pH 8.0, 0.1% SDS) with 1% Triton X-100 and 150 mM NaCl and incubated with 3μg ChIP-grade antibody at 4°C overnight with gentle shaking. 5% of input was stored prior to the de-crosslinking procedure. 30μl Protein-G magnetic beads (Thermo Fisher Scientific, 01134323) was used for subsequent pull-down of antibody-chromatin complex by incubatioin for 2 hours at 4°C with gentle shaking. The beads were washed with following buffers for 2 times each: IP buffer (0.1% SDS, 1% Triton X-100, 1mM EDTA, 150mM NaCl, 10mM Tris-HCl pH 8.0), High Salt Wash Buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 500mM NaCl, 20mM Tris-HCl pH 8.0), LiCl Wash Buffer (250mM LiCl, 1% NP-40, 2mM EDTA, 10mM Tris-HCl pH 8.0), and TE buffer+50mM NaCl (1mM EDTA, 50mM NaCl, 10mM Tris-HCl pH 8.0) 1 times. DNA was eluted with Elution Buffer (1% SDS, 10mM EDTA, 50mM Tris-HCl pH 8.0) at 65℃ for 30 minutes with shaking at 1,400 rpm. The supernatant was then collected and incubated at 65°C for overnight to reverse-crosslink the DNA. The day after, the enriched DNA was treated with 4 µL of 20 mg/mL RNase at 37 °C for 2 h and 4 µL of 20 mg/mL Proteinase K (MIKX, FZ690) at 55°C for 2h. The immunoprecipitation DNA was purified by phenol-chloroform-isoamyl alcohol (Solarbio, P1012-100), washed with ethanol, eluted in nuclease-free water, and used for subsequent experiments. The following antibodies were used in ChIP: H3K4me1 (Abcam, ab8895), H3K4me3 (Abcam, ab8580)
Sequencing libraries were constructed and sequenced by the Novogene Bioinformatics Institute (Novogene, Beijing, China).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
The quality controls of all reads were checked using FastQC (http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc/).
Filter sequencing adapters and low-quality reads using TrimGalore (v0.6.6).
Cleaned reads were aligned to the hg38 genome with bowtie2 (v 2.4.5)
Peaks were called using MACS2 (v 2.2.7.1)
Bigwig files adjusted by input data and enrichment profiles across genomic regions of interest were all generated using deeptools
Assembly: GRCh38/hg38
Supplementary files format and content: Bigwig file
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| Submission date |
Jan 10, 2023 |
| Last update date |
Jan 12, 2023 |
| Contact name |
Cao Qiang |
| E-mail(s) |
csqasds@gmail.com
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| Organization name |
Sun Yat-sen University
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| Department |
School of Medicine
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| Street address |
No. 66 Gongchang Road, Guangming District, Shenzhen
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| City |
Shenzhen |
| ZIP/Postal code |
518000 |
| Country |
China |
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| Platform ID |
GPL16791 |
| Series (2) |
| GSE222523 |
Pan-cancer analysis revealed H3K4me1 at bivalent promoters premarks DNA hypermethylation during tumor development and identified the regulatory role of DNA methylation in relation to histone modifications [ChIP-seq] |
| GSE222612 |
Pan-cancer analysis revealed H3K4me1 at bivalent promoters premarks DNA hypermethylation during tumor development and identified the regulatory role of DNA methylation in relation to histone modifications |
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| Relations |
| BioSample |
SAMN32658662 |
| SRA |
SRX18988632 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6925409_HCT116_WT_ChIPseq_H3K4me3_rep1_aln.bw |
333.7 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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