 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jul 15, 2018 |
| Title |
MBD3_f_-_IPS_Oct4_reanalyzed |
| Sample type |
SRA |
| |
|
| Source name |
MBD3_f_-_IPS_Oct4
|
| Organism |
Mus musculus |
| Characteristics |
genotype/variation: MBD3flox/- mouse Embryonic Fibroblast Cells (MEF) transgenic for DOX inducible OSKM reprogramming
stage in reprogramming: iPSC
target protein: Oct4
antibody: Santa Cruz sc5279(C-10)
|
| Treatment protocol |
DOX induction at day 0 at the relevant plate in final concentration of 2ug/ml
|
| Growth protocol |
ESCs, iPSCs, and 4-8 day DOX induced cells were expaned in N2B27 15% KSR 2i/LIF mouse ESC media; MEF cells in DMEM + 15% FBS
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Approximately 25*10^6 cells were cross-linked in formaldehyde (1% final concentration, 10 min at room temperature (RT)), and then quenched with glycine (5 min at RT). Fixed cells were lysed in 50mM HEPES KOH pH 7.5, 140mM NaCl, 1mM EDTA, 10% Glycerol, 0.5% NP-40 alternative, 0.25% Triton supplemented with protease inhibitor at 4°C (Roche, 04693159001), centrifuged at 950 x g for 10 min and re-suspended in 0.2% SDS, 10mM EDTA, 140mM NaCl and 10mM Tris-HCL. Cells were then fragmented with a Branson Sonifier (model S-450D) at -4°C to a size range between 200 and 800 bp, and precipitated by centrifugation. 10 ug of antibody were pre-bound by incubating with Protein-G Dynabeads (Invitrogen100-07D) in blocking buffer (PBS supplemented with 0.5% TWEEN and 0.5% BSA) for 2 hours at room temperature. Washed beads were added to the chromatin lysate, and then incubated overnight. Samples were washed 5 times with RIPA buffer, twice with RIPA buffer supplemented with 500 mM NaCl, twice with LiCl buffer (10 mM TE, 250mM LiCl, 0.5% NP-40, 0.5% DOC), once with TE (10Mm Tris-HCl pH 8.0, 1mM EDTA), and then eluted in 0.5% SDS, 300 mM NaCl, 5 mM EDTA, 10 mM Tris Hcl pH 8.0 at 65°C. Eluate was incubated in 65°C for 4 hours, and then treated sequentially with RNaseA (Roche, 11119915001) for 30 min and Proteinase K (NEB, P8102S) for two hours. DNA was purified with The Agencourt AMPure XP system (Beckman Coulter Genomics, A63881).
120ul SPRI AMPure XP beads (Agencourt) were added to the reverse-crosslinked samples, pipette-mixed 15 times and incubated for 2 minutes. Supernatant were separated from the beads using a magnet for 4 minutes. Beads were washed on the magnet with 70% ethanol and then air dried for 4 minutes. The DNA was eluted in 40 ul EB buffer (10 mM Tris-HCl pH 8.0) by pipette mixing 25 times. For the remainder of the library construction process (DNA end-repair, A-base addition, adaptor ligation and enrichment) a general SPRI cleanup involves addition of buffer containing 20% PEG and 2.5 M NaCl to the DNA reaction products (without moving them from their original well position). After thorough mixing and a 2-minute incubation at room temperature, plates are transferred to a magnet plate, incubated for 4 minutes and supernatant removed. Beads are then washed on the magnet with 150ul 70% ethanol and then air dried for 4 minutes. The DNA is eluted with 40ul of EB buffer by pipette mixing 25 times. Reagent kits are prepared in advance for all enzymatic steps (New England Biolabs). The DNA end-repair was performed by adding 27 µl of a master mix (17 µl master mix (5 ul T4 buffer, 5ul BSA-1mg/ml, 5ul ATP-10mM -2ul dNTPs 10 mM), 5 ul T4 PNK enzyme, 5 µl T4 polymerase (3 units) to each well. Samples were incubated in a thermal cycler at 12C for 15 min, 25C for 15 min, and finally cooled to 4C. The SPRI bead clean up method was used to purify the product (147 µl of 20% PEG, 2.5 M NaCl was added to each sample and eluted in 40 µl EB). The A-base addition was performed by adding 20 µl master mix (17 µl A-base add mix, 3 µl Klenow (3’->5’ exonuclease) to each well and incubated at 37C for 30 min. in a thermal cycler. SPRI bead clean up method was used to purify the product (132 µl of 20% PEG, 2.5 M NaCl was added to each sample and eluted in 19 µl EB). Adaptor ligation was performed by adding 34 µl of a master mix (29 µl 2x DNA ligase buffer, 5 µl DNA ligase) to each well. Finally 5 µl PE Indexed oligo adaptors (0.75 uM ) was added to each well and samples were incubated 25C for 15 min in a thermal cycler. SPRI bead clean up with size selection was used to purify the ligated products (15.5 µl of 20% PEG, 2.5 M NaCl was added to each sample and eluted in 40 µl EB). Finally, enrichment PCR was performed by adding 10 µl of a master mix (2 µl Forward/Reverse Index Primer, 0.5 µl dNTP mix, 5 µl 10x Pfu Ultra Buffer, 1 µl Pfu Ultra-II Fusion, 1.5 µl Nuclease free water) to each well. Plate was transferred to a thermal cycler and ran a Pfu amplification program at 95C for 2 min, 16 cycles of: 95C for 30 sec, 55C for 30 sec, 72C for 60 sec, and finally 72C for 10 min. The final SPRI clean up coupled to size selection was performed (35 µl SPRI beads was added to each sample and eluted in 40 µl). Approximately 5 picomoles of DNA library was then applied to each lane of the flow cell. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Illumina Hiseq2000 Analyzer following the manufacturer's protocols.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
GSM1631260
|
| Data processing |
We used Illumina bcl2fastq version 2.17.1.14 software to generate fastq files.
Reads were aligned to mouse mm10 reference genome using bowtie2 software, with default parameters.
For transcription factors, peaks were identified with MACS version 1.4.2-157. Whole-cell extract was used as control to define a background model. Duplicate reads aligned to the exact same location are excluded by MACS default configuration. Chromatin modification coverage in the promoters was calculated using in-house script. Shortly, promoters were defined as 1Kb region around the TSS, based on mm10 assembly. Each promoter was divided to 50bp size bins, and the coverage in each bin is estimated.
Genome_build: mm10
Supplementary_files_format_and_content: Peak files (bed format) and *prom_500.matrix.txt files. In the processed matrix file, for each promoter is the coverage in 20 bins, 50bp each.
|
| |
|
| Submission date |
Jan 03, 2018 |
| Last update date |
Jul 16, 2018 |
| Contact name |
Noa Novershtern |
| E-mail(s) |
noa.novershtern@weizmann.ac.il
|
| Organization name |
Weizmann Institute of Science
|
| Department |
Molecular Genetics
|
| Street address |
Weizmann Institute
|
| City |
Rehovot |
| ZIP/Postal code |
7610001 |
| Country |
Israel |
| |
|
| Platform ID |
GPL13112 |
| Series (2) |
| GSE102517 |
High-Resolution Dissection of Conducive Somatic Cell Reprogramming to Naïve Ground State Pluripotency in Mbd3/NuRD and Gatad2a/NuRD Depleted Systems [ChIP-Seq] |
| GSE102518 |
High-Resolution Dissection of Conducive Somatic Cell Reprogramming to Naïve Ground State Pluripotency in Mbd3/NuRD and Gatad2a/NuRD Depleted Systems |
|
| Relations |
| Reanalysis of |
GSM1631260 |
| BioSample |
SAMN08288653 |
| SRA |
SRX3528890 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2912463_4-Oct_iPS_peaks.GSM1631260.bed.gz |
105.4 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
|
|
|
|
 |
