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| Status |
Public on Jul 01, 2014 |
| Title |
AGS-H3K27me3-ChIP-BS-repeat1 |
| Sample type |
SRA |
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| Source name |
gastric adenocarcinoma cells
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| Organism |
Homo sapiens |
| Characteristics |
cell type: gastric adenocarcinoma cells
chip antibody: H3K27me3
cell line: AGS
chip antibody manufacturer: Millipore
chip antibody catalog #: 17-622
chip antibody lot #: DAM1739345
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| Growth protocol |
A lymphoblastoid cell line was generated from a male Han Chinese individual (YH), whose genome sequence was reported previously [28]. Two human gastric cancer cell lines (BGC-823 and AGS) were provided by Beijing Tumor Hospital; the HeLa cell line was purchased from the American Type Culture Collection (ATCC). Cells were cultured with RPMI1640 (Gibco C22400500BT) supplemented with 10% fetal bovine serum (Gibco 12657-029) in a humidified incubator with 5% CO2 at 37°C.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP-seq, approximately 5×106 cells were used. Proteins were cross-linked to DNA with 1% formaldehyde in 10 ml PBS at 37°C for 10 min, thenand then the cells were washed with pre-cooled PBS with 0.5% bovine serum and by PBS supplemented with protease inhibitor compound (PIC). The cells were collected by centrifugation at 850g for 3 min after each wash. The cells were resuspended in 200 μl ice-cold lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, plus PIC) and then thawed on ice for 10 min to allow lysis. The cell lysate was then sonicated for 180 s using a BioruptorTM 200 at the highest power (pulses: 30 s on/30 s off) to generate chromatin fragments between 100 and 700 bp. As input, one-tenth of the sonicated chromatin sample was removed. The remaining chromatin was immunoprecipitated in ChIP dilution buffer (1% Triton, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl) with 4 µg antibody against H3K4me3 (Millipore; 17-614) or H3K27me3 (Millipore; 17-622) that had been pre-incubated with protein A/G magnetic beads (Invitrogen; 10003D). After immunoprecipitation overnight, the beads were washed twice with each of the following buffers at 4°C: RIPA buffer (10 mM Tris, 1 mM EDTA, 0.1% SDS, 0.1% Na-deoxycholate, 0.1% Triton X-100); RIPA buffer plus 0.3 M NaCl; LiCl buffer (0.25 M LiCl, 0.5% NP-40, 0.5% Na-deoxycholate) and TE buffer. The beads were collected on a magnet for recovery. The bound DNA was then eluted from the beads with elution buffer（1% SDS, 0.1 M NaHCO3）at 65°C for 2-3 h.then sequenced on Illumina Hiseq2000 using a standard pair-end 50bp (PE50) sequencing protocol. ChIP-BS-seq:Ends of the immunoprecipitated DNA (60ng) from ChIP procedure were repaired in a 100μl reaction containing 1XT4 PNK buffer, 3 units T4 DNA polymerase (ENZYMATICS; P708), 0.5 unit klenow enzyme (ENZYMATICS; P7060), 10 units T4-PNK (ENZYMATICS; Y9040) and dNTP (0.125mM for each) for 30min at 20℃. Followed by treatment with 15 unit Klenow fragment (3’-5’exo, ENZYMATICS; P7010-LC) in a 50μl reaction containing 1Xblue buffer (ENZYMATICS; B011) and 0.2mM dATP at 37℃ for 30min to generate a protruding 3’A base. Then methylated pair-end adapters were ligated to the DNA fragment using 2400 unit of rapid T4 DNA ligase (ENZYMATICS; L6030-HC) at 37℃ for 15 min. After purification, 200ng exogenous λ-DNA fragments were added to the samples, and the sodium bisulfite conversion assay (ZYMO D5006) was performed, followed by 16 cycles of PCR amplification that was consisted of denaturation (94℃ for 15s), annealing (60℃ for 30s), extension (72℃ for 30s). Then, the PCR products were size-selected on 2% agarose gels, retaining 250-350 bp DNA fragments. The purified DNA (ChIP-BS libraries) was used for cluster generation and standard PE50 sequencing using Illumina Hiseq 2000.
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
stomach epithelial cells
AGS-H3K27me3-ChIP-BS-repeat1
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| Data processing |
Illumina BclConverter-1.9.0 software used for basecalling.
Adaptor sequences were removed, and low-quality sequence reads were trimmed. The clean reads of ChIP-seq reads were mapped to hg18 SOAP libraries using default parameters. For ChIP-BS library,We change C->T and G->A of reference respectively and build SOAP library using them, then, translate the C of sequences fq1 to T, G of fq2 to A, and mapped them with above libraries. (Lister R et al. (2009) Human DNA methylomes at base resolution show widespread epigenomic differences. Nature 462: 315-322.)
Using unique mapped reads, and after duplication reads (the pair end mapped reads share same postion only use the best mapped pair reads to eliminate the affect of PCR). then extract the methylation of CpG and the read densi ty of each sample. we further analysis the enrichment of each sample using RSEG software(0.4.4) the parameter using default with bin size 500bp and posterior cutoff 0.95.
Genome_build: hg18
Supplementary_files_format_and_content: bed files:ChIP information it contained chromsome,reads strart site,reads end sites; and for CpG Methylation of ChIP-BS it contained chromsome,CpG site,enriched reads number,and methylation of CpG; wig files include each samples ChIP signal on enriched sites.
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| Submission date |
Dec 21, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Desheng Gong |
| E-mail(s) |
gds19870718@163.com
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| Organization name |
Agricultural Genomes Institute at Shenzhen
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| Street address |
No.7 PengFei road
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| City |
Shenzhen |
| ZIP/Postal code |
518120 |
| Country |
China |
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| Platform ID |
GPL11154 |
| Series (2) |
| GSE43094 |
Direct ChIP-bisulfite sequencing reveals a role of H3K27me3 mediating aberrant hypermethylation of promoter CpG islands in cancer cells (ChIP-BS) |
| GSE43096 |
Direct ChIP-bisulfite sequencing reveals a role of H3K27me3 mediating aberrant hypermethylation of promoter CpG islands in cancer cells |
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| Relations |
| SRA |
SRX212306 |
| BioSample |
SAMN01840360 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1056537_AGS_H3K27me3-ChIP-BS-rep1.reads.bed.gz |
63.8 Mb |
(ftp)(http) |
BED |
| GSM1056537_AGS_H3K27me3-ChIP-BS-rep1.wig.gz |
19.2 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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