 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Mar 07, 2018 |
| Title |
OSKM Day 9 SMRT ChIP-seq |
| Sample type |
SRA |
| |
|
| Source name |
MEFs transfected with OSKM Day 9
|
| Organism |
Mus musculus |
| Characteristics |
day: 9
treatment: OSKM
presumed cell type: Intermediate pluripotent reprogramed cells
strain: C57BL/6J-OG2
antibody: anti-SMRT Abcam, ab24551 Lot# GR140820-1
|
| Treatment protocol |
15,000 MEFs were transducted once in 12-well plates using virus-containing supernatants generated by PlatE or HEK293T cells. After infection the medium was changed to mouse ESC serum-based medium (DMEM-High glucose supplemented with 15% FBS, non-essential amino acids, GlutaMAX, sodium-pyruvate, penicillin/streptomycin, 0.1 mM β-mercaptoethanol and 100 units/ml LIF or mouse ESC KSR medium (DMEM-High glucose supplemented with 15% Knockout Serum Replacement (Gibco), non-essential amino acids, GlutaMAX, sodium-pyruvate, penicillin/streptomycin, 0.1 mM β-mercaptoethanol, N2, 5 ng/ml basic fibroblast growth factor (bFGF) (Peprotech) and 100 units/ml LIF, and renewed daily. Cells were cross-linked in freshly prepared formaldehyde solution (1% final concentration for 10 minutes at room temperature), and then quenched with 125 mM glycine (5 minutes at room temperature). Fixed cells were washed with PBS, harvested, flash-frozen in liquid nitrogen and store at -80°C for further use.
|
| Growth protocol |
Maintenance of mouse ESCs and fully reprogramed iPSCs was conducted in chemically defined N2B27-based medium: DMEM/F12 (HyClone) and Neurobasal (Gibco) mixed 1:1, supplemented with N2 (Invitrogene), B27 (Invitrogene), non-essential amino acids, GlutaMAX, sodium-pyruvate (Gibco), penicillin/streptomycin (HyClone), 0.1 mM β-mercaptoethanol (Sigma), 100 units/ml leukemia inhibitory factor (LIF) (Millipore), small-molecule inhibitors CHIR99021 (3 μM, Selleck), and PD0325901 (1 μM, Selleck)
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were lysed in nuclei extraction buffer (50 mM HEPES KOH (pH 7.5), 140 mM NaCl, 1m M EDTA, 10% glycerol, 0.5% NP-40 alternative, 0.25% Triton X-100) supplemented with protease inhibitor cocktail (Roche) for 10 minutes at 4 °C. Cell pellets were resuspended in protein extraction buffer (10 mM Tris-HCl (pH 8.0), 200 mM NaCl, 1 mM EDTA and 0.5 mM EGTA) supplemented with protease inhibitor cocktail and incubated for 10 minutes at room temperature. For sonication, pellets were resuspended in sonication buffer (10 mM Tris-HCl (pH 8.0), 0.5 mM EGTA, 1% Triton X-100, protease inhibitor cocktail) and then fragmented with a Bioruptor (Diagenode) sonicator at 4°C for 30 cycles using high amplitude and 30 seconds ON and 30 seconds OFF cycles to produce size ranges between 200 and 500 base pairs. 2 mg of each antibody was prebound by incubating with Protein A+G Dynabeads (Invitrogen 100-07D) in blocking buffer (PBS supplemented with 0.5% TWEEN) for 6 hours at 4°C. Washed beads were added to the chromatin lysate and incubated overnight. Samples were washed twice with low salt washing buffer (20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X-100), twice with high salt washing buffer (20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X-100) twice with LiCl buffer (10 mM Tris-HCl, 250mM LiCl, 1mM EDTA, 1% NP-40, 1% Na-deoxycholate), twice with TE (10 mM Tris-HCl pH 8.0, 1mM EDTA), supplemented with 50 mM NaCl and eluted in elution buffer (50 mM Tris-HCl, 10 mM EDTA and 1% SDS). Eluates were incubated at 65°C for 20 minutes, followed by de-crosslinking at 65°C for 6-15 hours. Samples were diluted in TE buffer and then treated with RNaseA (Roche) for 60 minutes at 37°C, followed by incubation with proteinase K (Sigma) for 45 minutes at 56°C. DNA was purified with phenol:chloroform:isoamyl alcohol. The following antibodies were used for ChIP experiments: anti-NCoR (ABE251, Millipore), anti-SMRT (ab24551, Abcam), anti-H3K27ac (ab4729, Abcam).
Standard Illumina protocols
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
Reads were aligned to the mouse mm10 genome using bowtie2 v2.2.0
Peaks were discovered using MACS 1.4 (NCoR/SMRT)
Peaks were discovered using Dfilter (H3K27ac)
Genome_build: mm10
Supplementary_files_format_and_content: Processed data files are peak bed files and bigWig tracks
|
| |
|
| Submission date |
Jul 10, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Andrew P Hutchins |
| E-mail(s) |
andrewh@sustech.edu.cn
|
| Organization name |
Southern University of Science and Technology
|
| Department |
Bioinformatics
|
| Lab |
Bioinformatics and Genomics
|
| Street address |
1088 Xueyuan Rd
|
| City |
Shenzhen |
| ZIP/Postal code |
518055 |
| Country |
China |
| |
|
| Platform ID |
GPL13112 |
| Series (2) |
| GSE70736 |
NCoR/SMRT co-repressors cooperate with c-MYC to create an epigenetic barrier to somatic cell reprogramming [ChIP-Seq] |
| GSE70740 |
NCoR/SMRT co-repressors cooperate with c-MYC to create an epigenetic barrier to somatic cell reprogramming |
|
| Relations |
| BioSample |
SAMN03854026 |
| SRA |
SRX1091751 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1817010_oskm_d9_smrt.bw |
139.3 Mb |
(ftp)(http) |
BW |
| GSM1817010_oskm_d9_smrt_peaks.bed.gz |
67.0 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
