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| Status |
Public on May 03, 2012 |
| Title |
hMeDIP-Seq-induced |
| Sample type |
SRA |
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| Source name |
NTera2 D1 cell line
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| Organism |
Homo sapiens |
| Characteristics |
cell line: NTera2 D1 cell line
developmental stage: differentiated
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| Treatment protocol |
NT2 cells were induced to differentiate with 10 µM all-trans retinoic acid (Sigma). Medium containing RA was changed every 2 days.
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| Growth protocol |
NT2 cells were maintained in Dulbecco's Modified Eagle Medium (D-MEM, with l-glutamine, 4500 mg/l d-glucose, without sodium pyruvat; Gibco) supplemented with 10% fetal bovine serum and 200 U/ml penicillin and 200 µg/ml streptomycin (all from Gibco) in a humidified atmosphere of 5% CO2 in air.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was purified from untreated and RA-treated NT2 cells as described (Mohn et al., 2009). Fifty micrograms of genomic DNA for each sample was sonicated to 300bp fragments using a Covaris S220 (Covaris, Massachusetts, USA) according to manufactures instructions (5 cycles, each 60s: 10%; intensity=5, dust=100). Sheared DNA was ethanol precipitated, washed with 70% ethanol, and dissolved in TE buffer (pH8.0). Illumina adapters were ligated before (h)MeDIP as described before (Xu et al., 2011). Briefly, End-It DNA End-repair kit (Epicentre, Madison, USA) was used for 1h incubation at RT on sheared DNA in 150 µl reaction volume consisting of 12 µg DNA diluted to 102 μl with ddH2O, 15μl of 10x End-it buffer, 15μl of 2.5 mM dNTP mix, 15μl of 10 mM ATP and 3μl of End-it enzyme. DNA was purified with QiaQuick PCR purification kit (Qiagen) and diluted to 122.5μl with ddH2O. Then 15μl of NEB buffer 2, 3.5μl of 10mM ATP and 9μl of 5U/μl Klenow exo- (NEB) were added. After incubating at 37°C for 50min, the “A” base added DNA was purified with QiaQuick PCR purification kit, diluted to 24μl with ddH2O, and mixed with 50μl of 2x NEB quick ligation buffer, 20 μl of barcoded Illumina adapter and 6μl of quick T4 DNA ligase (NEB). DNA was purified with QiaQuick PCR purification kit after incubating at RT for 20 min. Ligation efficiency was analyzed by PCR on 10ng of final ligated DNA with Illumina sequencing primers and ran on a 1% agarose. Five micrograms of adapter ligated DNA was used for (h)MeDIP as described above. Immunoprecipitated DNA was finally resolved in 20µl of water and all MeDIP samples were pooled to create the MeDIP and hMeDIP libraries, respectively. Both libraries were purified on a e-Gel (Invitrogen) and the 300bp bands (containing roughly 200bp fragments of adapter-ligated immunopurified DNA) were extracted. Then, libraries were amplified using Illumina-Enrichment Primers for 12 cycles and purified using Agencourt AMPure XP beads (Beckman Coulter, Brea, USA) according to manufactures instructions. DNA concentrations were measured using a Agilent High Sensitivity DNA Assay (Agilent Technologies, Santa Clara, USA) and 7 pico mol were used for cluster PCR and substantial 50bp single-end sequencing on a HiSeq 2000 (Illumina).
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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 |
All processing steps of the sequencing data were carried out using the short read analysis pipeline shore (Ossowski et al., 2008). Reads were trimmed by removing stretches of bases having a quality score <30 at the ends of the reads. The trimmed reads were mapped to the most recent human genome assembly hg19 using the mapping program bowtie (Langmead et al., 2009). After the mapping duplicate reads were removed and the position-wise coverage of the HOXA cluster by sequencing reads was determined.
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| Submission date |
Oct 20, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
michael thomas bocker |
| Organization name |
DKFZ Heidelberg
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| Street address |
Im Neuenheimer Feld
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| City |
heidelberg |
| ZIP/Postal code |
69120 |
| Country |
Germany |
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| Platform ID |
GPL11154 |
| Series (2) |
| GSE33129 |
Hydroxylation of 5-methylcytosine by TET2 maintains the active state of the mammalian HOXA cluster (MeDIP-Seq) |
| GSE33130 |
Hydroxylation of 5-methylcytosine by TET2 maintains the active state of the mammalian HOXA cluster |
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| Relations |
| SRA |
SRX101671 |
| BioSample |
SAMN00739934 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM820541_hmC_14dRA.sam.gz |
3.8 Gb |
(ftp)(http) |
SAM |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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