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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Oct 11, 2013 |
| Title |
resting_Smc3_CHIP-seq |
| Sample type |
SRA |
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| Source name |
CD43- B cells extracted from spleen
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
chip antibody: Smc3 (Abcam ref: ab9263 lot: GR81379-1)
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| Treatment protocol |
Cells were fixed with 1% paraformaldehyde at 37°C for 10 minutes followed by quenching with 0.125 M glycine (final concentration). After lysis, chromatin was sonicated to generate 0.2–0.5 kb DNA fragments using a Covaris sonicator (Covaris). After dilution in ChIP buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1, and 167 mM NaCl), chromatin was precleared by rotating 1 hour at 4°C with 400µl of dynabeads protein A (Invitrogen). Dynabeads were coupled with specific or control antibodies for 1h at room temperature. Pre-cleared chromatin was precipitated overnight with the dynabeads coupled with the antibodies (10 × 106 cell equivalents were used as input, and 100 × 106 cell equivalents were used for ChIP) and processed according to the Millipore protocol.
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| Growth protocol |
Resting B cells were isolated from the spleen of 8-12 week-old wild-type mice (C57BL/6J) with anti-CD43 Microbeads (anti-Ly48; Miltenyi Biotech) and were cultured for 60 h with LPS (50 μg/ml; Sigma Aldrich) and IL-4 (5 ng/ml; Sigma Aldrich).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP-seq libraries were prepared using ChIP-Seq Sample Prep Kit (#IP-102-1001, Illumina) following the manufacturer's protocol with some modifications. Briefly, 10 ng of ChIP enriched DNA or control DNA were end repaired using T4 DNA polymerase, Klenow DNA polymerase and T4 PNK. A single ‘A’ nucleotide was added to the 3’ ends of the blunt DNA fragments with a Klenow fragment (3' to 5'exo minus). The ends of the DNA fragments were ligated to double stranded adapters using T4 DNA Ligase. The ligated products were enriched by PCR (30 sec at 98°C; [10 sec at 98°C, 30 sec at 65°C, 30 sec at 72°C] x 14 cycles; 5 min at 72°C) and then purified using Agencourt AMPure XP beads (#A63881, Beckman). DNA library size selection was performed by excising the 250-350 bp fragments from a 2% agarose gel followed by purification using a QIAquick Gel Extraction Kit (#28906, Qiagen).
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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 |
Illumina Casava1.8 software used for basecalling.
Sequence reads were mapped to reference genome mm9/NCBI37 using Bowtie v0.12.7 with the following parameters -m 1 --strata --best.
Genome_build: mm9
Supplementary_files_format_and_content: Wig files were generated using with an in-house script (Variable step, span=25, reads were elongated to 200b)
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| Submission date |
Jan 17, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Anne-Sophie THOMAS |
| E-mail(s) |
[email protected]
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| Organization name |
IGBMC
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| Lab |
REINA
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| Street address |
1 rue Laurent Fries
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| City |
Illkirch |
| State/province |
67 |
| ZIP/Postal code |
67400 |
| Country |
France |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE43594 |
Genome-wide localization of Smc1, Smc3 and CTCF in mouse B cells. |
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| Relations |
| SRA |
SRX218289 |
| BioSample |
SAMN01889198 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1066045_wigs_for_resting_Smc3_2.wig.gz |
246.5 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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