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| Status |
Public on Mar 01, 2016 |
| Title |
CTCF_ChIP_MEF_p3_2 |
| Sample type |
SRA |
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| Source name |
MEF_p3_2
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| Organism |
Mus musculus |
| Characteristics |
cell type: MEF derived p3 iPS
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| Growth protocol |
ESCs and iPSCs were cultured on Mitomycin-C treated MEFs in KO-DMEM (Invitrogen) supplemented with 1% nonessential amino acids (Invitrogen), 0.1 mM β-mercaptoethanol (Invitrogen), 1,000 U/ml LIF (Millipore) and 15% fetal bovine serum (FBS, Invitrogen) (ESC medium). Naïve ESCs were cultured in serum-free N2B27 medium on gelatin-coated dishes. N2B27 medium (500ml) was generated by inclusion of the following: 240ml DMEM/F12 (Invitrogen), 240ml Neurobasal (Invitrogen), 5ml N2 supplement (Invitrogen), 10ml B27 supplement (Invitrogen), 1,000 U/ml LIF (Millipore), 1% nonessential amino acids (Invitrogen), 0.1mM β-mercaptoethanol (Invitrogen), and the small molecules PD0325901 (Stemgent, 1μM) and CHIR (Stemgent, 3μM). CD19 + pre-B cells and Mac1 + macrophages were isolated from bone marrow with monoclonal antibodies to CD19 and Mac-1 (BD Pharmingen) respectively, using MACS sorting (Miltenyi Biotech). B cells were grown in RPMI medium with 10% FBS and 10ng/ml IL-7 (Peprotech); macrophages in DMEM with 10% FBS and 10ng/ml each of CSF1 and IL-3 (Peprotech). MEFs were established from day 13.5 mouseembryos and cultured in DMEM containing 10% FBS. NSC were isolated and cultured aspreviously described (Di Stefano et al., 2009). All media were supplemented with L-glutamineand penicillin/streptomycin (GIBCO).
Reprogramming experiments with B cells were performed as previously described (Di Stefano et al., 2014); with MEFs, macrophages and NSCs were conducted by plating 100.000 cells/well on gelatinized plates seeded with irradiated MEFs, using ESC medium supplemented with 2 μg/ml of doxycycline. For the isolation of iPSC lines, doxycycline was washed out after 15 days of reprogramming and colonies with ESC-like morphology were picked at 20 days before further passaging. iPSC lines were expanded for an additional 9 days (3 passages) to obtain P3 iPS cell lines or for 20 passages to obtain P20 iPS cell lines.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP experiments were performed as described previously (van Oevelen et al., 2008) using an antibody against H3K27ac (ab4729, Abcam) and CTCF (Millipore, 07-729). DNA libraries were prepared using Illumina's reagents and instructions.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
ChIP
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| Data processing |
Fastq files were aligned to the mouse genome (mm9) using bwa aln.
Peak calling was performed using MACS2 (callpeak), with --extsize 180 and -g mm.
Genome_build: mm9
Supplementary_files_format_and_content: narrowPeak
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| Submission date |
Jan 04, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Elzo de Wit |
| Phone |
+31 30 2121 800
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| Organization name |
Hubrecht Institute
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| Street address |
Uppsalalaan 8
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| City |
Utrecht |
| ZIP/Postal code |
3584 CT |
| Country |
Netherlands |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE76478 |
Cell-of-origin specific 3D genome structure acquired during somatic cell reprogramming [ChIP-Seq] |
| GSE76481 |
Cell-of-origin specific 3D genome structure acquired during somatic cell reprogramming |
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| Relations |
| BioSample |
SAMN04382645 |
| SRA |
SRX1513739 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2026241_CTCF_MEF_p3_2_peaks.narrowPeak.gz |
1.6 Mb |
(ftp)(http) |
NARROWPEAK |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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