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| Status |
Public on Dec 03, 2012 |
| Title |
pro-B input control |
| Sample type |
SRA |
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| Source name |
Rag1 -/- pro-B cells
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
genotype/variation: Rag1 -/-
age: 5- to 8-wk-old
tissue: pro-B cells
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| Growth protocol |
Bone marrow cells were isolated from 5- to 8-wk-old Rag1−/− mice and were enriched for CD19+ cells with MACS microbeads.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
B6 Rag1−/− pro-B cells were crosslinked with 1% formaldehyde for 10 minutes at room temperature. Subsequently, the lysates were sonicated using a Diagenode Biorupter. The chromatin solution was precleared with salmon sperm DNA-protein A agarose beads. The lysate was immunoprecipitated with antibodies against CTCF (Millipore 07-729) . Immune complexes were isolated with protein A agarose beads. Following elution, chromatin-antibody complexes and input DNA were reverse crosslinked by heating at 65°C overnight. The DNA was purified using the Qiagen DNA purification kit.
The sequencing libraries were prepared using 10 ng double-stranded cDNA prepared by ChIP (pull-down and input). ChIP DNA samples ends were repaired using recommended Illumina protocol including T4 DNA polymerase, Klenow Large Fragment, and T4 polynucleotide kinase. DNA products were purified using DNA Clean&Concentrator™-5 Kit (Zymo Research). Next, DNA ends were A-tailed with Klenow (3’→5’ exo-) at 37C for 30 min. DNA products were again purified using the DNA Clean&Concentrator™-5 Kit. Next, Illumina Paired End-adapter oligonucleotides, at 0.33 μM concentration, were ligated to the A-tailed cDNA ends with T4 DNA ligase. DNA products were purified using DNA Clean&Concentrator™-5 Kit. The DNA library products were then separated on a 2% agarose gel and products corresponding to a size of approximately 200-250 bases were removed from the gel and cleaned using the Agencourt SPRI system (Beckman). The DNA material was PCR amplified with Phusion™ Polymerase (Finnzymes) with 0.6 μM PCR primers PE 1.0 and PE 2.0 (Illumina) for 15 cycles. The amplified DNA products were further purified on 2% agarose gel, excised, and isolated again using Zymoclean™Gel DNA recovery kit. The purified DNA library was quantitated using the Qubit quantitation platform (Invitrogen) and sized using the 2100 Bioanalyzer (Agilent). DNA products were then denatured in 0.1N NaOH and diluted to a final concentration of 7 s pM before being loaded onto the Illumina paired-end flow-cell for massively parallel sequencing by synthesis on the Illumina HiSeq2000.
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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 |
Data filtering by duplicate read removal.
Mapping with bowtie. Settings -q -y -a -v 2 -p 8 -m 3 --best --strata
Filtering for uniquely mapped reads as well as reads mapped to multiple locations within the IgH, TCR, and LINE repeats.
Binning of reads in 25 bp windows.
Significant bins determined by dynamic windows utilizing Poisson distribution where the null distribution is based upon the the greater of the entire genome, 1kb, 5kb, and 10kb windows as baseline.
Genome_build: mm9
Supplementary_files_format_and_content: prob-ctcf.wig - CTCF reads with input control reads subtracted. prob-ctcf.peaks.txt - significant peaks at 1e-5 threshold of significance.
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| Submission date |
Oct 22, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Ali Torkamani |
| E-mail(s) |
atorkama@scripps.edu
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| Organization name |
TSRI
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| Street address |
3344 North Torrey Pines Court Suite 300
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| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92037 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (2) |
| GSE41741 |
ChIP-seq analysis of CTCF binding sites in lymphocyte subsets [pro-B cells] |
| GSE41743 |
ChIP-seq analysis of CTCF binding sites in lymphocyte subsets |
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| Relations |
| SRA |
SRX199986 |
| BioSample |
SAMN01774231 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data not provided for this record |
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