|
| Status |
Public on Apr 13, 2011 |
| Title |
mC_DnmtTKO |
| Sample type |
SRA |
| |
|
| Source name |
embryonic stem cells
|
| Organism |
Mus musculus |
| Characteristics |
chip or dip: DIP
cell type: embryonic stem cells
genotype/variation: Dnmt tripple knock out
chip/dip antibody: methycytosine
|
| Treatment protocol |
Cells were either untransfected or transfected with a scrambled or Tet1 specific shRNA
|
| Growth protocol |
Low passage (p17) E14TG2a.4 feeder independent ES cells were cultured on 0.1% gelatin-coated plates in Glasgow medium (Sigma) supplemented with glutamine (Gibco), nonessential amino acids (Gibco), sodium pyruvate (Gibco), 50 ìM ßmercaptoethanol, and 15% foetal bovine serum (HyClone) in the presence of LIF. Recombinant lentiviruses encoding TET1 shRNA were produced by standard methods employing co-transfection of PLKO.1 shRNA and packaging vectors in 293FT cells. shRNA transduced ES cells were selected 36 hours post transduction with 2 ìg per ml of puromycin for 72 hours.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin immunoprecipitation assays (ChIP) were performed and analysed as previously described {Pasini et al. (2010). Nature. 2010 Mar 11;464(7286):306-10. ES cell DNA was sonicated to an average size between 300-600 bp. Adaptor-ligated libraries for hmC or mC DNA immunoprecipitation assays (hm-DIP/me-DIP) were constructed using the NEBNext™ DNA Sample Prep Master Mix, NEB combined with Illumina adaptors. hme/me-DIP assays were performed as described by {Weber et al., Nat Genet. 2005 Aug;37(8):853-62} using one µg of denatured sonicated or adaptor-ligated DNA in 100 ul of binding buffer and 0.1-4 µg of affinity purified rabbit hmC antibody or monoclonal mC antibody (Eurogentec BI-MECY-0500). The samples were incubated for four hours at 4ºC before addition of 10 µl of anti-rabbit/mouse Dynabeads (InVitrogen). After two hours of incubation, the samples were washed four times and bound DNA was eluted by incubation for one hour at 55 ºC in 100 ml of 50 mM Tris-HCl, 10 mM EDTA, 0.5% SDS and 20 mg proteinase K. The DNA was purified using a Qiaquick PCR purification kit (Qiagen) and amplified by 16 cycles of PCR. For ChIP-seq analysis, the DNA obtained from the ChIP assays were adaptor-ligated and amplified using a kit from Illumina (IP-102-1001). The amplified DNA from hme/me-DIP or ChIP-seq experiments was analyzed by Solexa/Illumina high-throughput sequencing. The tags were mapped to the mouse genome (assembly mm9) with the Bowtie alignment tool and the Solexa Analysis Pipeline.
DNA was adaptor-ligated and amplified using a kit from Illumina (IP-102-1001). The amplified DNA from hme/me-DIP or ChIP-seq experiments was analyzed by Solexa/Illumina high-throughput sequencing. The tags were mapped to the mouse genome (assembly mm9) with the Bowtie alignment tool26 and the Solexa Analysis Pipeline. To avoid any PCR bias we allowed only one count (match) per chromosomal position thus eliminating spurious spikes. Peak detection, binding and gene annotation analysis were performed using the CisGenome program22 at an FDR cut-off value <0.1 or <0.01 as indicated in the text. IgG was used for normalization
|
| |
|
| Library strategy |
MeDIP-Seq |
| Library source |
genomic |
| Library selection |
5-methylcytidine antibody |
| Instrument model |
Illumina Genome Analyzer |
| |
|
| Data processing |
Preprocessing: Sequenced adapters and low quality reads were removed. Alignment: Tags were mapped to the mouse genome (assembly mm9) with the Bowtie alignment tool with parameters: -t --best -k 1 -y -p 4 --solexa1.3-quals --chunkmbs 512 -n 2 -l 28 -e 120 and the Solexa Analysis Pipeline. To avoid any PCR bias we allowed only one count (match) per chromosomal position thus eliminating spurious spikes. Peak detection, binding and gene annotation analysis were performed using the Cisgenome program.
|
| |
|
| Submission date |
Oct 20, 2010 |
| Last update date |
May 15, 2019 |
| Contact name |
Kristine Williams |
| E-mail(s) |
kristine.williams@bric.dk
|
| Organization name |
University of Copenhagen
|
| Department |
BRIC
|
| Lab |
Helin
|
| Street address |
Ole Maaløes vej 5
|
| City |
Copenhagen |
| ZIP/Postal code |
2200 |
| Country |
Denmark |
| |
|
| Platform ID |
GPL9185 |
| Series (2) |
| GSE24841 |
Tet1 and hydroxymethylcytosine in transcription and DNA methylation fidelity (ChIP/DIP-Seq data) |
| GSE24843 |
Tet1 and hydroxymethylcytosine in transcription and DNA methylation fidelity |
|
| Relations |
| SRA |
SRX029873 |
| BioSample |
SAMN00117120 |
| Named Annotation |
GSM611206_mC_DnmtTKO.bowtie.bed.gz |
