 |
 |
GEO help: Mouse over screen elements for information. |
|
| 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 |
| E-mail(s) |
matthieu.gerard@cea.fr
|
| 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 |
|
| Relations |
| BioSample |
SAMN04271234 |
| SRA |
SRX1433587 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1941477_MNase-seqs_shRNA-Ep400_transfection_replicate_1_B00H5QG_5_1_C7BP7ACXX.IND117.bw |
527.9 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
