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| Status |
Public on Dec 20, 2012 |
| Title |
H9_medip |
| Sample type |
SRA |
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| Source name |
embryonic stem cells
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| Organism |
Homo sapiens |
| Characteristics |
cell type: embryonic stem cells
passages: 29
cell line: H9
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| Growth protocol |
For maintenance of H9 hESCs, MEF feeder layer was obtained from CF-1 mouse embryos and cultured in MEF medium, which is composed of 90% DMEM (GIBCO 12430) supplemented with 10% FBS (Biochrom S0615). Before freezing cells, MEFs were cultured for 3h at 37℃ in mitomycin C (Sigma M0503) medium that is composed of MEF medium supplemented with 10 ug/ml mitomycin C. For culturing H9 ES cells, MEF-conditioned medium was produced by conditioning MEFs for at least 24 hours in the medium composed of DMEM/F12 (GIBCO 11330) supplemented with 20% knockout serum replacement (GIBCO 10828), 2 mM L-glutamine (GIBCO 25030), 2 mM nonessential amino acids (GIBCO 11140), 0.1 mM 2-mercaptoethanol (GIBCO 21985-023), and 4 ng/ml recombinant human fibroblast growth factor-basic (bFGF; GIBCO 13256-029).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted from H9 hESCs using QIAamp DNA Blood Mini Kit (QIAGEN)
Prior to immunoprecipitation, 5 µg of genomic DNA was sonicated to a mean size of 200bp fragments, followed with end repair, <A> base addition and adapter ligation steps according to Illumina Paired-End protocol. MeDIP-seq and hMeDIP-seq libraries were constructed on 500 ng of adapter ligated DNA using the Magnetic Methylated DNA Immunoprecipitation kit (Diagenode) and hMeDIP 10rxns kit (Active Motif) following the manufacturer’s instructions, respectively. After immunoprecipitation, the captured products were then eluted and purified on a single ZYMO DNA Clean & Concentrator-5 column following the manufacturer’s instructions. Finally, PCR amplification was carried out using platinum pfx DNA polymerase (Invitrogen) with a thermal cycling program of 94°C 2 min, 12cycles of 94 °C 15s, 60°C 30s, 72°C 30 then prolonging with 5min at 72°C and products could be hold at 12°C. Amplification quality and quantity were evaluated by 2100 Analyzer and QPCR. The libraries were sequenced using Illumina HiSeq 2000 according to the manufacturer’s instructions.
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| Library strategy |
MeDIP-Seq |
| Library source |
genomic |
| Library selection |
5-methylcytidine antibody |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Illumina BclConverter-1.9.0 software used for basecalling.
Low-quality reads were omitted and the clean reads were aligned to the UCSC human reference genome (hg19) using SOAP2 (Version 2.21) 24. Mismatches of no more than two bases were allowed in the alignment.
Region between pair-end reads mapped to the genome was regarded as an enriched fragment. If only one of the pair-end reads mapped to the genome, the read was extended to 200bps and the extended read was regarded as an enriched fragment. Based on these enriched fragments, wig files were generated by calculating the average fragment numbers in each 100bp window on the genome.
Genome_build: hg19
Supplementary_files_format_and_content: H9_medip.wig.gz Average MeDIP enriched fragments number in each 100bp window on the genome
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| Submission date |
Sep 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) |
| GSE41070 |
Integrated detection of both 5-mC and 5-hmC by high-throughput tag sequencing technology highlights methylation reprogramming of bivalent genes during cellular differentiation [MeDIP_hMeDIP] |
| GSE41071 |
Integrated detection of both 5-mC and 5-hmC by high-throughput tag sequencing technology highlights methylation reprogramming of bivalent genes during cellular differentiation |
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| Relations |
| SRA |
SRX189181 |
| BioSample |
SAMN01728930 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1008198_H9_medip.wig.gz |
32.9 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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