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| Status |
Public on Dec 01, 2011 |
| Title |
Mbd3 rep1 ChIP |
| Sample type |
SRA |
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| Source name |
Mbd3 ChIP
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| Organism |
Mus musculus |
| Characteristics |
genotype/variation: WT
cell type: embryonic stem (ES) cells
chip antibody: Mbd3
chip antibody vendor & cat.#: Abcam (ab3755)
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| Treatment protocol |
In one 10 cm plate, 1.4X10^6 cells were transfected with 2 micrograms of esiRNA using Lipofectamine 2000. After 12-15 hours, the media was replaced and cells were cultured until 48 hours post-transfection.
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| Growth protocol |
Feeder-free growth on gelatin coated dishes in standatd ES medium: KO DMEM + 10% FBS, glutamine, LIF, non-essential amino acids, beta-mercaptoethanol.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP material was gel purified (100 to 800bp in size) from a 2% TAE agarose gel using MiniElute columns QiaQuick (Qiagen). Gel purified DNA fragments were blunt ended and phosphorylated with an EPICENTRE End-it-Repair kit (1X buffer, 0.25mM dNTPs,1mM ATP, 1ul/50ul reaction of Enzyme mix) for 1hr at RT and cleaned up with Qiagen MiniElute spin columns. Adenosine nucleotide overhangs were added using EPICENTRE exo- Klenow for 45min at RT (with 0.2mM dATP). Illumina genome sequencing adaptors were then ligated using the EPICENTRE Fast-Link ligation kit: 11.5ul A tailed DNA eluted from a MinElute column was mixed with 1.5ul 10X ligation buffer, 0.75ul 10mMATP, 0.5ul Illumina DNA adaptors and 1ul ligase. The reaction was incubated for 1hr at RT and subsequently supplemented with 7.5 ul water, 1ul 10X buffer, 0.5ul 10mM ATP and 1ul ligase, and incubated overnight at 16C. The ligation reaction was cleaned up with MiniElute columns (with an additional wash step to eliminate all the excess adaptors) and the adaptor ligated fragments were amplified by PCR as follows:0.75 ul of each Illumina genomic DNA sequencing primers, 6ul 10xPfx buffer 1.8ul 10mM dNTPs, 1.2ul 50mM MgSO4 and 1ul Pfx DNA polymerase (Invitrogen) were added to 30ul DNA template in a 100ul reaction. The cycling parameters were: 1. 94C 2'; 2. 94C 15''; 3. 65C 1'; 4. 68C 30''; 5. repeat from step 2 16 times; 6. 68C 5'. The PCR product (250 to 450bp in size) was gel purified from a 2% TAE agarose gel using the QiaQuick columns (Qiagen). Gel purified fragments were finally precipitated with Sodium acetate and ethanol and pellets were resuspended (25nM final concentration) in TE buffer.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer II |
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| Data processing |
The alignments are done using Bowtie allowing only one mismatch and unique mapper (*.mapped.txt). The aggregation signals around each TSS (+/- 4000bp) are tabulated in flat format (*.txt)
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| Submission date |
Aug 26, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Jui-Hung Hung |
| E-mail(s) |
juihnughung@gmail.com
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| Organization name |
Boston University
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| Street address |
24 cummington st
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| City |
Boston |
| ZIP/Postal code |
02215 |
| Country |
USA |
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| Platform ID |
GPL9250 |
| Series (1) |
| GSE31690 |
A key role for Mbd3 in 5-hydroxymethylcytosine-dependent gene regulation in embryonic stem cells |
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| Relations |
| SRA |
SRX095348 |
| BioSample |
SAMN00715042 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM786608_Mbd3IP_rep1.raw.mapped.txt.gz |
45.6 Mb |
(ftp)(http) |
TXT |
| GSM786608_Mbd3IP_rep1.raw.txt.gz |
2.2 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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