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| Status |
Public on Oct 15, 2013 |
| Title |
H3K4me2 ChIP-Seq C57BL6/J 100 ng/ml Kdo2-LipidA 1 hr |
| Sample type |
SRA |
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| Source name |
peritoneal thioglycolate-elicited macrophages
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL6/J
treatment: 100 ng/ml Kdo2-LipidA 1 hr
chip (antibody): H3K4me2 (Vendor: Millipore, cat# 07-030, lot# DAM1724042)
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| Treatment protocol |
Cells were treated by replacing the seeding media with fresh, warm media ("notx") or fresh, warm media containing 100 ng/ml KDO2-lipid A (KLA) for the indicated times.
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| Growth protocol |
Peritoneal macrophages were harvested by peritoneal lavage with 20 ml ice-cold PBS 4 days after peritoneal injection of 3 ml thioglycollate broth. Peritoneal cells seeded in 10% fetal calf serum (FCS)/RPMI-1640 containing 100 U/ml penicillin/streptomycin in tissue culture-treated petri dishes overnight. Non-adherent cells were washed off with room temperature PBS.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP-Seq: Media was decanted and cells were fixed at room temperature with either 1% formaldehyde/PBS for 10 minutes (for PU.1, C/EBPα ChIPs) or 2 mM disuccinimidylglutarate (DSG, Pierce)/10% DMSO/PBS for 30 minutes followed by 1% formaldehyde/PBS for another 15 minutes. After quenching the reaction by adding glycine to 0.125 M, cells were scraped into the supernatant using a rubber policeman, pelleted, washed twice with PBS and snap-frozen in dry-ice/methanol. ChIPs for PU.1 and C/EBPα were performed exactly as described previously (Heinz, S. et al. Molecular Cell 38, 576-589 (2010)). Briefly, 7e6-20e6 cells were incubated for 5 min in 1 ml 0.5% IGEPAL CA-630-containing 10 mM HEPES pH 7.9/85 mM KCl/1 mM EDTA on ice, centrifuged, sonicated on wet ice with a Misonix 3000 sonicator in 500 µl 1% SDS/0.5% EmpigenBB/50 mM Tris pH7.4/10 mM EDTA/1x protease inhibitor cocktail/1 mM PMSF for 6 cycles of 10s/30s break, lysates were cleared for 5 min, 20000 x g, 4°C, and the supernatant diluted with 1.5 volumes dilution buffer (0.5 % Triton X-100/20 mM Tris pH 7.4/100 mM NaCl/2 mM EDTA). Diluted chromatin was precleared for 1 h with 120 µl 50% slurry of CL-4B sepharose blocked (washed twice with TE buffer, blocked for 60 min in 0.5% BSA/20 µg/ml glycogen/TE, washed twice with TE, resuspended in original volume of TE), then immunoprecipitated overnight by adding 2.5 µg antibody, and capture with 50 µl protein A-sepharose (GE Healthcare Life Sciences, blocked as above, but overnight). Immunoprecipitates were washed twice each with ice-cold wash buffer I (150 mM NaCl/1% Triton X-100/0.1% SDS), II (450 mM NaCl/1% Triton X-100) , III (250 LiCl/1% IGEPAL CA-630/1% Deoxycholate) and TE, and eluted with 1% SDS/TE at room temperature. After addition of NaCl to 300 mM final Na+ concentration, crosslinks were reversed overnight in a hot air oven, RNA digested for 2 h at 37°C with 0.33 mg/ml RNase A, proteins digested for 2 h at 50°C with 0.5 mg/ml proteinase K, and DNA extracted using Qiagen QiaQuick columns. The H3K27Ac ChIPs were performed in the presence of 1mM (C57BL/6J and BALB/cJ) or 5 mM (CB6F1J) butyric acid in all buffers, using sepharose as described above for the C57BL/6J and BALB/c experiments, while for the CB6F1J experiment 5 µg antibody pre-bound to 50 µl Protein A Dynabeads (Life Technologies) were used, and the preclearing step was omitted. For p65, IP conditions were identical to the ones described before, except that pre-clearing was omitted, and the ChIP was performed with 5 µg antibody pre-bound to 50 µl Protein A Dynabeads for 30 minutes in 0.5% BSA, beads were washed thrice with WB I and thrice with WBIII containg only 0.7% deoxycholate, and a final wash with TE/50 mM NaCl was added before elution. ChIPs for H3K4me2 were performed on MNase-digested chromatin as described previously (Barski et al. Cell 129, 823-837 (2007)) on 20e6 cells digested in two 10e6 cell batches with 5 and 10 U MNase (Worthington) for 5 min at 37°C, s adding stop buffer containing 10 mM each of EDTA/EGTA, brief sonication in a Bioruptor 200, dialysis against two changes of RIPA buffer, and overnight IP with antibody-bound, block protein A-Dynabeads (Life Technologies) at 4°C. After two washes each with RIPA buffer, 0.3 M NaCl/RIPA buffer, 0.5% deoxycholate/0.5% IGEPAL CA-630-containing buffer and TE, DNA was eluted with 0.3% SDS/TE and 0.9 mg/ml proteinase K overnight at 65°C overnight, and DNA was isolated by phenol/chloroform extraction and ethanol precipitation. To control for open chromatin and library biases, input chromatin libraries after sonication were sequenced for each strain, crosslinking condition and ChIP lysis protocol. Sequencing libraries were prepared by blunting, A-tailing, adapter ligation as previously described (Heinz, S. et al. Molecular Cell 38, 576-589 (2010)) using barcoded adapters (NextFlex, Bioo Scientific). Libraries were PCR-amplified for 12-14 cycles, size selected by gel extraction and sequenced on a Hi-Seq 2000 (Illumina) for 51 cycles.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
Mnase-treated genomic DNA
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| Data processing |
For ChIP-Seq, RNA-Seq and GRO-Seq samples, reads were aligned to the mm9 genome as well as BALB/cJ contigs using bowtie (v0.12.7), keeping only reads that mapped best to a single, unique location in both genomes, while allowing 3 mismatches in the first 28 bases, and at least 5 high-quality mismatches overall. The NOD PU.1 notx ChIP-Seq sample was only mapped to mm9 according to the same criteria. To identify allele-specific reads in CB6F1J, the initial mapping to strain-specific regions in both genomes defined in the parental inbred strains was performed with the leftmost 33 bases allowing no mismatch, and reads that mapped to both genomes were discarded. Aligned read files were analyzed with HOMER (http://biowhat.ucsd.edu/homer/) to find peaks, calculate RPKM from the gene bodies of RefSeq genes, perform motif- and other analyses in the study. Genetic variation analyses were performed using custom R scripts.
Genome_build: mm9, BALB/cJ de novo assemblies (Keane, T. M. et al., Nature 477, 289-294 (2011), the initial published version) were downloaded from the Sanger Institute website: http://www.sanger.ac.uk/resources/mouse/genomes/
Supplementary_files_format_and_content: Processed files include BED-like files (ChIP-Seq peak positions), rpkm text files (RNA-Seq, GRO-Seq expression data). All genomic coordinates are relative to mm9 (NCBI 37) mouse assembly.
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| Submission date |
Apr 30, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Casey Romanoski |
| Organization name |
UCSD
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| Street address |
9500 Gliman Dr.
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| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92093 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE46494 |
Natural genetic variation perturbs collaborative transcription factor binding required for enhancer selection and function |
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| Relations |
| BioSample |
SAMN02087240 |
| SRA |
SRX272799 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1131247_H3K4me2.KLA.C57BL6.regions.txt.gz |
1.4 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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