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Sample GSM2026249 Query DataSets for GSM2026249
Status Public on Mar 01, 2016
Title CTCF_ChIP_preB_p3_2
Sample type SRA
Source name preB_p3_2
Organism Mus musculus
Characteristics cell type: preB derived p3 iPS
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.
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.
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina NextSeq 500
Description ChIP
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
Submission date Jan 04, 2016
Last update date May 15, 2019
Contact name Elzo de Wit
Phone +31 30 2121 800
Organization name Hubrecht Institute
Street address Uppsalalaan 8
City Utrecht
ZIP/Postal code 3584 CT
Country Netherlands
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
BioSample SAMN04382648
SRA SRX1513747

Supplementary file Size Download File type/resource
GSM2026249_CTCF_preB_p3_2_peaks.narrowPeak.gz 1.8 Mb (ftp)(http) NARROWPEAK
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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