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Sample GSM1941477 Query DataSets for GSM1941477
Status Public on Jan 26, 2016
Title Ep400 knockdown MNase-seq replicate 1
Sample type SRA
Source name Ep400 knockdown MNase-seq, ESC
Organism Mus musculus
Characteristics strain: 46C (129/Ola strain)
cell line: ESC line 46C
transfection: shRNA vector targeting Ep400, replicate 1
Treatment protocol described in the SAMPLES section (experimental conditions)
Growth protocol Embryonic stem cells were cultured at 37°C, 5% CO2, on mitomycin C-inactivated mouse embryonic fibroblasts, in DMEM (Sigma) with 15% foetal bovine serum and leukemia inhibitory factor (LIF).
Extracted molecule genomic DNA
Extraction protocol 1 million cells were fixed 10 min in culture medium containing 1% formaldehyde and ollected in 15 mM Tris-HCl PH7.5, 0.3 M sucrose, 60 mM KCl, 15 mM NaCl, 5mM MgCl2, 0.1 mM EGTA, 0.4% Igepal CA-630 (Sigma). We next added 2 mM CaCl2 and 4 units of MNase, and incubated 10 min at 37°C. MNase digestion was stopped by adding 10 mM EDTA (final concentration), and storing on ice. Cells were then disrupted by 15 passages through a 25G needle, followed by a 10 min centrifugation at 18,000 g. The supernatant was collected and incubated 1h at 65°C with 15 mg of RNase A. We next added 10 mg of proteinase K, adjusted each sample to 0.1% SDS (final concentration) and incubated 2 h at 55°C. NaCl concentration was then adjusted to 200 mM and the samples were incubated overnight at 65°C for crosslink reversal. DNA was purified from each sample by phenol-chloroform extraction followed by ethanol precipitation.
20 ng of purified DNA was used for library preparation according to manufacturer's instructions, using Ultralow ovation library system, Nugen. Following end-repair and adapter ligation, fragments were size-selected onto an agarose gel in order to purify genomic DNA fragments between ~ 60 and 220 bp.
Library strategy MNase-Seq
Library source genomic
Library selection MNase
Instrument model Illumina HiSeq 2000
Description MNase-seq
Data processing Basecalling was performed with RTA 1.18.64. Bcl conversions into Fastq and demultiplexing were performed using bcl2fastq version 1.8.3, Sequenced reads were trimmed for adaptor sequence and masked for low-complexity/low-quality sequence with Trimmomatic 0.32, then mapped to mm9 whole genome using bowtie v0.12.7 with parameters -a -m1 --best --strata -v2 p3
Genome_build: mm9
Supplementary_files_format_and_content: wig files were generated using WigMaker 3 with default parameters; Scores represent the maximum number of reads overlapping in a 25 bp bin
Supplementary_files_format_and_content: BIGWIG files were generated with UCSC tools (v4) using WigToBigWig program
Submission date Nov 16, 2015
Last update date May 15, 2019
Contact name Matthieu GERARD
Organization name CEA
Department I2BC
Lab Mammalian Epigenomics
Street address SBIGeM bldg 142
City Gif-sur-Yvette
ZIP/Postal code 91191
Country France
Platform ID GPL13112
Series (1)
GSE64825 Genome-wide distribution and function of ATP-dependent chromatin remodelers in embryonic stem cells
BioSample SAMN04271234
SRA SRX1433587

Supplementary file Size Download File type/resource 527.9 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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