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| Status |
Public on Dec 05, 2013 |
| Title |
5hmC_TCBQ |
| Sample type |
SRA |
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| Source name |
MRC-5_TCBQ_5hmC
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| Organism |
Homo sapiens |
| Characteristics |
cell line: MRC-5
cell type: human fetal lung fibroblast Cell
enrichment method: The Hydroxymethyl Collector™ Kit
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| Treatment protocol |
TCBQ powder was dissolved in DMSO with the concentration of 20mM for stock. MRC-5 cells with about 50% confluence were treated with TCBQ or DMSO diluted in culture media to the final concentration of 20uM for 24 hours.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted from the harvested cells using a Genomic DNA Purification Kit (Promega, Madison, WI, USA), according to the manufacturer’s instructions. 0.1 mM DFO was added in the processes of DNA extraction and purification.
Genomic DNA from TCBQ or DMSO treated MRC-5 cells was purified by using Wizard genomic DNA purification kit (Promega) with additional Proteinase K treatment and rehydrated in 10 mM Tris (pH 8.0). Genomic DNA samples were further sonicated into short fragments (about 300bp) by using Covaris DNA shearing (Covaris S2 SonoLAB Single) with microTUBEs according to manufacturer’s instructions. Sonicated DNA was then purified with phenol/chloroform/isoamyl alcohol (25:24:1) precipitation. 5hmC was pulled down as described previously (Song et al., 2011). After purification, 10ng of the pull-down 5hmC containing DNA was end-repaired, adenylated, ligated to adapters and single-end sequenced on an HiSeq 2000 system (Illumina) according to the manufacturer’s recommendations for Illumina ChIP-Seq to identify peak enrichment
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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 |
ChIP-seq reads were aligned to the hg19 genome assembly using BWA version 0.5.9-r16
Peaks were called using MACS version 1.4.2 with default parameter
We aligned those high quality reads to the masked Homo sapiens reference genome (hg19) genome and junction sequences using BWA 0.5.8 software allowing up to four mismatches. All the uniquely mapped reads were used to define gene expression level based on our developed method. We modified the RPKM (measured in reads per kilobase of exon per million mapped sequence reads, which is a normalized measure of exonic read density) calculation described by Mortazavi and colleagues to include junction fragments. By including reads that map to junction fragments, we ensure that genes with many introns are not under represented in the RPKM estimate. To examine differential expression, we used a MARS (MA-plot-based method with Random Sampling model) method to identify differentially expressed genes which described in DEGseq. For each gene, the P-value and Q-value were computed, and the fold changes were also calculated. Those genes with p < 0.001 and q-value < 0.05 were defined as DEGs.
Genome_build: hg19
Supplementary_files_format_and_content: The files are generated by MACS version 1.4.2, including chr, start, end, length, summit, tags, -10*log10(pvalue),fold_enrichment. The file of differitially expressed genes includes Gene, GeneNames, value1, value2, log2(Fold_change), log2(Fold_change) normalized, z-score, p-value, q-value(Benjamini et al. 1995), q-value(Storey et al. 2003), Signature(p-value < 0.001).
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| Submission date |
Feb 21, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Zechen Chong |
| Organization name |
Beijing Institute of Genomics
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| Street address |
NO. 7 Beitucheng West Road
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| City |
Beijing |
| ZIP/Postal code |
100029 |
| Country |
China |
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| Platform ID |
GPL11154 |
| Series (1) |
| GSE44457 |
Cellular generation of 5-Hydroxymethylcytosine by redox-active chemicals via an unprecedent non-enzymatic mechanism |
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| Relations |
| SRA |
SRX243743 |
| BioSample |
SAMN01923905 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1085704_5hmc_TCBQ_peaks.txt.gz |
1.4 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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