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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Dec 16, 2015 |
| Title |
DNASE_TKO_2 |
| Sample type |
SRA |
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| Source name |
embryonic stem cells
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| Organism |
Mus musculus |
| Characteristics |
strain: 159 (mixed 129-C57Bl/6)
genotype: DNMT TKO
growth condition: serum
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| Growth protocol |
Mouse embryonic stem cells were cultivated without feeders on 0.2% gelatine-coated dishes in DMEM, supplemented with 15% fetal calf serum, 1× non-essential amino acids, 2 mM L-glutamine, LIF and 0.001% β-mercaptoethanol (37°C, 7% CO2).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNase treatment was performed essentially as previously described, with some modifications (John et al. Curr Prot Mol Biol 2013). Briefly, intact nuclei were extracted using 0.03% NP-40 in an isotonic buffer. After NP-40 removal, batches of 5 million nuclei were incubated for 4 min at 37°C with a range of DNase I (DPRF, Worthington) concentrations in the presence of Ca2+. The digestion was stopped by addition of EDTA/SDS and the samples were treated with proteinase K and RNase A. Phenol-chloroform extracted DNA was separated on a 5-30% sucrose gradient by ultracentrifugation for 24 h and fractionated with a Gilson fraction Collector FC 203B. Fractions were precipitated with ethanol and resuspended in TE buffer. Both successful digestion and size separation were verified by agarose gel electrophoresis. Low-coverage sequencing of a bar-coded pool of samples derived from different fractions of the sucrose gradient and treated with different DNase concentrations was used to select the sample with the highest information content.
Library Construction Protocol: Libraries for DNase-seq were prepared essentially according to standard Illumina protocols, using 40 ng of the precipitated fractions of the sucrose gradient as starting material. To reduce amplification bias, end-repaired, A-tailed and adapter ligated DNA was amplified in 6 cycles of PCR with KAPA HiFi Hot Start polymerase. Adapter dimers were subsequently removed with Agencourt AMPure XP beads (Beckman Coulter). Quality of the libraries and size distribution was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies). Sequencing was performed on an Illumina HiSeq 2500 machine (50 bp read length, single end) according to Illumina standards.
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| Library strategy |
DNase-Hypersensitivity |
| Library source |
genomic |
| Library selection |
DNAse |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Basecalling was performed with the Illumina RTA 1.17.21.3 software with default parameters
Fastq files were generated with the Illumina bcl2fastq-1.8.4 software with options --use-bases-mask Y50n,I6n --fastq-cluster-count 100000000 --no-eamss
Raw reads were trimmed of illumina adaptors. Read mapping was performed using Bowtie version 1.0.0 with parameters -v 3 -m 1 --best --strata.
Bigwig files were generated by using the 1bp 5'-end of the reads and calculating for each genomic position the read density normalized to one million reads in the library.
Genome_build: mm9
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| Submission date |
Apr 14, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Anais Flore Bardet |
| Organization name |
IGBMC
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| Street address |
1 rue Laurent Fries
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| City |
Illkirch |
| ZIP/Postal code |
67404 |
| Country |
France |
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| Platform ID |
GPL17021 |
| Series (1) |
| GSE67867 |
Competition between DNA methylation and transcription factors determines binding of NRF1 |
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| Relations |
| BioSample |
SAMN03482409 |
| SRA |
SRX994802 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1657367_DNASE_TKO_2.bw |
423.3 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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