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| Status |
Public on Jun 13, 2018 |
| Title |
MNase-seq_Ssrp1-KD_rep1,rep2 |
| Sample type |
SRA |
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| Source name |
mESCs
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| Organism |
Mus musculus |
| Characteristics |
cell line: E14 cell line
treatment: Ssrp1 knock down
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| Growth protocol |
The E14 cell line (mESCs) was cultured at 37 °C, 7.5% CO2, on 0.1% gelatin coated plates, in DMEM + GlutaMax™ (Gibco) with 15% fetal bovine serum (Gibco), MEM non- essential amino acids (Gibco), penicillin/streptomycin (Gibco), 550 µM 2-mercaptoethanol (Gibco), and 10 ng/ml of leukaemia inhibitory factor (LIF) (eBioscience).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Biological replicates were obtained from two independent transfection experiments for each shRNA vector. Briefly, ~5 million cells were crosslinked with 1% formaldehyde for 20 min followed by quenching for 5 min with the addition of glycine to a final concentration of 0.125 M. After washing with PBS buffer, cells were collected and lysed in Cell Lysis buffer (5 mM Tris pH8.0, 85 mM KCl, 0.5% NP40 ) with proteinase inhibitors (10 µl/mL Phenylmethylsulfonyl fluoride (PMSF), 1 µl/mL Leupeptin and 1 µl/mL Pepstatin). Nuclei were gathered by centrifugation (5000 rpm for 2 min) and were treated with 10 Kunitz Units of micrococcal nuclease (NEB, #M0247S) per 10^6 cells for 5 min at 37°C. The reaction was stopped with the addition of 60 µl 50 mM EDTA, 25 µl 5 M NaCl, 15 µl 20% NP-40 and incubated on a rotator for 1h at room temperature to release soluble nucleosomes. Samples were centrifuged for 5 min at 10,000 g and supernatant was transferred to a new tube. Samples were reverse-crosslinked by incubating overnight at 65°C with 0.5% SDS and proteinase K. Following phenol-chloroform extraction and ethanol precipitation, DNA was incubated at 37°C for 4h with RNAse (Sigma). All the samples were run in a 2% agarose gel and fragments <200 bp were extracted and purified using the NucleoSpin® Gel and PCR Clean-up kit (Macherey-Nagel) according to the manufacturer’s instructions.
Purified DNA (500 ng) was used for library preparation as described previously (Tessarz, et al). The only difference was the PCR amplification step where we used the same conditions as mentioned in Henikoff et al but with only three amplification cycles.
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| Library strategy |
MNase-Seq |
| Library source |
genomic |
| Library selection |
MNase |
| Instrument model |
Illumina HiSeq 3000 |
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| Data processing |
MNase-seq BAM files were merged per condition, converted to bigwig, binned (1 bp), smoothed (20-bp window), and normalised to x1 sequencing depth. Mononucleosomes were selected according to fragment length (135-170 bp). Duplicated reads were removed.
Genome_build: mm10
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| Submission date |
Dec 05, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Peter Tessarz |
| E-mail(s) |
ptessarz@age.mpg.de
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| Organization name |
Max Planck Institute
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| Street address |
Joseph-Stelzman-Str 9b
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| City |
Cologne |
| State/province |
Nordrhein-Westfalen |
| ZIP/Postal code |
50931 |
| Country |
Germany |
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| Platform ID |
GPL21493 |
| Series (1) |
| GSE90906 |
Transcriptional repression by FACT is linked to regulation of chromatin accessibility at the promoter of ES cells |
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| Relations |
| BioSample |
SAMN06111826 |
| SRA |
SRX2396612 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2417334_KD_1.135_20.bw |
9.1 Gb |
(ftp)(http) |
BW |
| GSM2417334_KD_2.135_20.bw |
9.9 Gb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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