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| Status |
Public on Mar 01, 2016 |
| Title |
NSC_iPS_p20_1 Hi-C |
| Sample type |
SRA |
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| Source name |
NSC iPS p20 1
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| Organism |
Mus musculus |
| Characteristics |
cell type: NSC iPS p20
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| Treatment protocol |
cells are cross linked using 2% formaldehyde for 10minutes at room temperature in 10%FCS/PBS
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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 |
Cells are cross linked using 2% formaldehyde for 10min at room temperature in PBS 10%FCS, nuclei are isolated, after which chromatin is digested with DpnII subsequently ligated. After reversal of the cross links the DNA is purified (3C template).
The 3C template is fragmented and TruSeq libraries are prepared according to the vendor protocol.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
TruSeq libraries prepared from 3C template
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| Data processing |
Library strategy: HiC
Forward and reverse (R1,R2) sequencing files are seperately aligned to the mouse genome (mm9) using bwa mem. Duplicates are removed and mapped pairs are filtered using the hicup_filter from the HiCUP package. Pairs that fall within 1kb of each other are removed from the sample as they likely represent non-cut genomic DNA fragments.
Genome_build: mm9
Supplementary_files_format_and_content: Hi-C summary files contain the genomic position and strand of the two pairs of a Hi-C ligation
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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 |
GPL17021 |
| Series (2) |
| GSE76479 |
Cell-of-origin specific 3D genome structure acquired during somatic cell reprogramming [HiC-Seq] |
| GSE76481 |
Cell-of-origin specific 3D genome structure acquired during somatic cell reprogramming |
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| Relations |
| BioSample |
SAMN04382662 |
| SRA |
SRX1513830 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2026279_NSC_iPS_p20_1_summary.txt.gz |
166.0 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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