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| Status |
Public on Jul 13, 2020 |
| Title |
ChIPseq of SOX2 in iPSCs |
| Sample type |
SRA |
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| Source name |
iPSCs
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| Organism |
Mus musculus |
| Characteristics |
cell type: induced pluripotent stem cells
antibody: Anti-mSox2 (2748S, Cell Signaling)
treatment: NONE
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| Treatment protocol |
~500,000 of C57BL/6 early-passage MEFs (Passage 1-3) were co-transduced overnight with lenti-supernatants of FUW-M2rtTA and TetO-FUW-OSKM followed by medium change [MEF medium (High-Glucose DMEM, 15% FBS, Glutamax, P/S, NEAA)] and recovery of the culture for an additional day. The transduced cells were expanded and then passaged in 0.1% gelatin pre-coated 6 cm dishes. The induction of OSKM overexpression was initiated 24 hours later by the addition of doxycycline (DOX) at 2 g/ml final concentration. Half of the dishes were treated with doxycycline (+DOX), while the other half was left untreated (-DOX). Cells were harvested at selected time points during reprogramming course
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| Growth protocol |
MEFs were cultured and reprogrammed as described (Hussein SMI et al., 2014 & Polo JM et al., 2012)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
MEFs undergoing reprogramming and control cells (naïve MEFs, mESCs, miPSCs) were cultured under optimum conditions and were fixed at a high-confluence stage at room temperature for 10 minutes using 1% formaldehyde in fixing buffer, followed by quenching with 0.125M glycine at room temperature for 5 minutes. Upon extensive washes, cells were resuspended in Lysis [50mM Hepes (pH 7.9), 140mM NaCl, 1mM EDTA, 10% Glycerol, 0.5% NP-40, 0.25% Triton X-100] and then in sonication buffer [0.1% SDS, 1mM EDTA, 10mM Tris (pH 8.1)]. Chromatin shearing was carried out in the Covaris S2 sonicator using the Covaris TC12x12mm tubes (Tube AFA Fiber and Cap, Covaris) for 12 min (200 cycles per burst) allowing the shearing of chromatin within a range of 250-500 bps DNA fragments. Triton X-100 and NaCl were then added in the sheared chromatin to final concentrations of 1% and 150 mM, respectively. The chromatin was then centrifuged and the harvested supernatants were filtered throughout a 0.2 m syringe. ChIPs were carried out by incubating 75 μg of chromatin (corresponding to approximately 5x107 cells) with 2-10μg of antibody per ChIP reaction [Anti-mOct4 (19857, Abcam), Anti-mSox2 (2748S, Cell Signaling), Anti-mKlf4 (H-180, SC-20691, Santa-Cruz), Anti-mc-Myc (N-262, SC-764, Santa-Cruz) and rabbit IgG (crude serum)] overnight at 4°C. Next, Protein G-Dynabeads (10004D, Thermo Fisher Scientific) pre-equilibrated in IP buffer [0.1% SDS, 1mM EDTA, 10mM Tris (pH 8.1), 1% Triton X-100, 150mM NaCl], were incubated with the chromatin-antibody solution in an orbital mixer at 4°C for 2 hours. The recovered resin was subsequently washed with low and high salt buffers and LiCl buffer and the captured chromatin fragments were subjected to Proteinase K (03115828001, Roche Life Sciences) digestion at 50°C for 15 minutes, followed by overnight incubation with RNase A at 65°C. All DNA present in each sample was purified with AMPure XP beads and eluted in TE buffer.
Next Generation Sequencing (NGS) libraries were prepared using 1-15 ng of ChIP DNA and TruSeq adapters. DNA was blunt ended by End Repair Enzyme Mix (T4 DNA Polymerase, Klenow Fragment, T4 DNA Polynucleotide kinase) followed by “A” tailing of 3’ ends and ligation with the annealed TruSeq Adapters (Illumina). Conversion of the Y-shaped adapters to dsDNA occurred prior to the library size selection through 2.5% Metaphor/SeaKem LE (3:1 ratio) agarose gel electrophoresis. Each library was purified using miniElute columns (Qiagen) and then subjected to pre-amplification. The final quantification of DNA libraries was carried out according to the Quantification Standards of Illumina on an Agilent Technologies 2100 Bioanalyzer (Agilent Technologies).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Alignment was performed with Bowtie (1.0.0)
Filtering of low quality reads and multi-mapped reads was done with samtools and rmdup command
MACS 1.4.2 was used for peak calling
Genome_build: MM9 for mouse
Supplementary_files_format_and_content: bed files for OSKM binding
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| Submission date |
Jul 11, 2020 |
| Last update date |
Sep 12, 2024 |
| Contact name |
Dimitris Valakos |
| Organization name |
Biomedical Reasearch Foundation Academy of Athens
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| Department |
Center of Basic Research
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| Lab |
Molecular Biology Lab
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| Street address |
Soranou Ephessiou 4
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| City |
Athens |
| ZIP/Postal code |
11527 |
| Country |
Greece |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE114581 |
Construction of a dynamic gene regulatory network required for cellular reprogramming |
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| Relations |
| BioSample |
SAMN15512887 |
| SRA |
SRX8713913 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4667686_slop_im.bed.gz |
64.8 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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