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| Status |
Public on Feb 11, 2011 |
| Title |
Mature B cell CTCF |
| Sample type |
SRA |
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| Source name |
Ctcf ChIP DNA from Mature B cells
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6J
cell type: Mature B cell
genotype: wt
antibody: CTCF
antibody vendor: Milipore
antibody lot#: DAM1472197
antibody catalog#: 07-729
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| Treatment protocol |
5x10E6 cells were fixed with 1% formaldehyde in culture medium for 10 min at room temperature followed by quenching with 0.125 M glycine for 5 min. The cells were washed twice with ice-cold PBS and lysed in 1 ml of lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris pH8.0) for at least 30 min on ice. The crosslinked chromatin was sheared to an average size of 500 bp by ten 30-second pulses using a BiorupterTM sonicator (Diagenode). The lysate was then centrifuged to remove cell debris, and the chromatin was quantified in a UV spectrophotometer. The chromatin (400 µg) was diluted 10-fold in dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA 16.7 mM Tris pH8.10, 167 mM NaCl) and pre-cleared with 25 µl protein A sepharose beads (pre-absorbed with salmon sperm DNA and BSA) for 2 h at 4C. The chromatin supernatant was then incubated overnight with 5 µg of purified antibody at 4C. After incubation with protein A sepharose beads (50 µl) for 2h at 4C, the immune complexes were collected by centrifugation and washed with the following buffers each for 10 min at 4C; RIPA buffer (150 mM NaCl, 50 mM Tris pH8.0, 0.1% SDS), high-salt buffer(500 mM NaCl, 50 mM Tris pH8.0, 0.1% SDS, 1% NP40) LiCl buffer (250 mM LiCl, 50 mM Tris pH8.0, 0.5% Na deoxycholate, 1% NP40) and 2x TE (20 mM Tris pH8.0, 2 mM EDTA). The protein-DNA complexes were eluted from the beads in 450 µl elution buffer (1% SDS, 100 mM NaHCO3) at 55C for 2 h followed by the addition of proteinaseK to 500 µg/ml and overnight incubaton at 65C. Genomic DNA was isolated from the precipitated material as well as from the sheared chromatin input (1/100 of the material used for ChIP) by phenol extraction and ethanol precipitation.
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| Growth protocol |
Cells were cultured for 5 days.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Lysates were clarified from sonicated nuclei and TF-DNA complexes were isolated with antibody. Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer |
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| Description |
Chromatin IP against CTCF
Sample ID: 8310
average insert size: 307 nt
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| Data processing |
Illumina GA pipeline v1.4
Alignment: Sequence reads were obtained and mapped to the mouse genome (mm8, ncbi36) using bowtie 0.12.2 ( -v 2 --best --strata --tryhard -m 1 --solexa1.3-quals --chunkmbs 256)
Peaks: Peak detection was performed with the Model-based Analysis of ChIP-Seq (MACS) program (http://liulab.dfci.harvard.edu/MACS/) version 1.6.3.1 using default parameters
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| Submission date |
Feb 10, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Meinrad Busslinger |
| E-mail(s) |
busslinger@imp.ac.at
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| Phone |
00431-79730
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| Organization name |
Instutute of Molecular Pathology
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| Street address |
Dr. Bohrgasse 7
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| City |
Vienna |
| ZIP/Postal code |
1030 |
| Country |
Austria |
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| Platform ID |
GPL9185 |
| Series (2) |
| GSE27214 |
IGH analysis in pro-B cells with H3K4me3, H3K4me2, H3K9ac, H3K27me3, Pax5 and CTCF [ChIP-Seq] |
| GSE27215 |
IGH analysis in pro-B cells with H3K4me3, H3K4me2, H3K9ac, H3K27me3, Pax5 and CTCF |
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| Relations |
| SRA |
SRX041880 |
| BioSample |
SAMN00210882 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM672402_WT.Mature_B.CTCF-ab.30Y72AAXX.8310.6.p.bed.gz |
112.4 Mb |
(ftp)(http) |
BED |
| GSM672402_ctcf.matBcell.8310.32.1e-5_peaks.bed.gz |
528.2 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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