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| Status |
Public on Nov 23, 2015 |
| Title |
H3K4me3_Trim33-/-_0h |
| Sample type |
SRA |
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| Source name |
bone marrow derived macrophages
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| Organism |
Mus musculus |
| Characteristics |
cell type: bone marrow derived macrophages
strain: C57BL/6
genotype: Trim33-/-
chip antibody: H3K4me3 (07-473, Millipore, lot: JBC1888194)
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| Treatment protocol |
On day 7, macrophages were treated for 4h, 12h and 24h with 100ng/ml LPS (Sigma) in IMDM supplemented with 2.5% FCS. Cells were cross-linked with 1% formaldehyde for 10 min at 37°C, lysed in SDS lysis buffer [50 mM Tris pH8, 10 mM EDTA, 1% SDS, protease inhibitor cocktail (Roche)] and sonicated (cycle conditions: 15sec ON/30sec OFF, 15 min; Bioruptor, Diagenode) to obtain DNA fragments with an average length of 200-600 bp. Supernatant was diluted 10 times in IP dilution buffer [16.7 mM Tris pH8, 167 mM NaCl, 1.2 mM EDTA, 1.1% Triton X100, 0.01% SDS, protease inhibitor cocktail (Roche)] and immunoprecipitations were performed using 5μg of specific antibodies. Immunoprecipitated chromatin was collected using protein A agarose/Salmon Sperm DNA beads (Millipore) and, after washing and elution, reverse cross- linking was carried out with 0.2M NaCl at 65°C overnight. The chromatin was then digested by 20 μg of Proteinase K (Invitrogen) for 1h at 45°C and isolated by phenol- chloroform extraction.
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| Growth protocol |
Bone marrow cells were cultured in IMDM supplemented with 10% FCS (Invitrogen) 10μM thioglycerol (Sigma) and 25ng/ml mouse CSF1 (Milteny Biotech).
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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 Genome Analyzer IIx |
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| Data processing |
Illumina Casava 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 22, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
federica ferri |
| E-mail(s) |
federica.ferri@cea.fr
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| Organization name |
cea
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| Street address |
18 route du panorama
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| City |
Fontenay aux roses |
| ZIP/Postal code |
92265 |
| Country |
France |
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| Platform ID |
GPL11002 |
| Series (1) |
| GSE43654 |
Dynamics of TRIM33 binding in macrophages and effects of TRIM33 deletion on chromatin state during activation of primary macrophages. |
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| Relations |
| SRA |
SRX218754 |
| BioSample |
SAMN01893834 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1067644_wigs_for_H3K4me3_Trim33-_0h.wig.gz |
88.9 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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