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Status |
Public on Dec 31, 2020 |
Title |
GAR2107: Raw264.7_Setd1aDel/+_DexLPS_aH3K4me3_rep3 |
Sample type |
SRA |
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Source name |
Raw264.7
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Organism |
Mus musculus |
Characteristics |
cell type: Raw264.7 strain: Balb/c Sex: male genotype: Setd1aDel/+ spike-in: Drosophila melanogaster treatment: 16h 1uM Dex, 3h 100ng/ul LPS fixation: 10min 1% FA antibody: rabbit monoclonal a-H3K4me3 (05-745R, Millipore)
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Treatment protocol |
Raw264.7 cells were either treated with 1 uM dexamethason (Dex, Sigma #D4902) or 0.1% EtOH (=vehicle) for 16 h followed by 3h treatment with 100 ng/ul LPS (Sigma #L2630).
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Growth protocol |
Raw264.7 cells were kept at subconfluent conditions in DMEM (Gibco) supplemented with 10% FBS (heat inactivated, Sigma) and 1% penicillin/streptomycin (Gibco). Cells were cultured at 5% CO2 at 37°C in humified incubators. Medium was changed every other day.
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Extracted molecule |
genomic DNA |
Extraction protocol |
For ChIP-Seq, 40 mio cells per sample or 20 mio cells in case of H3K4me1/me2/me3 were fixed as mentioned in the sample section either for 30 min in 2 mM DSG (#C1104 ProteoChem) and 10 min 1% MeOH-free formaldehye (#2890, ThermoFisher) or 10 min 1% formaldehyde at room temperature. Remaining formaldehyde was quenched with 150 uM glycin, cells collected and washed 2x in ice-cold D-PBS. Pellets were stored at -80°C until ChIP was performed. For ChIP-Seq nuclei were extracted in Fast-IP buffer (150 mM NaCl, 5 mM EDTA (pH=7.5), 5 mM Tris (pH=7.5), 1% Triton X-100, 0.5% NP40), chromatin sonicated in shearing buffer (1% SDS, 10 uM EDTA (pH=8),50 mM Tris (pH=8)) using the Bioruptor Pico (Diagenode) for 10-15 cycles (30s on/off) at high settings. Sonicated chromatin was diluted 1:10 in dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 uM EDTA (pH= 8), 16.7 uM Tris (pH=8), 167 mM NaCl) and the immunoprecipitation against the protein of interest was performed over night at 4°C. Antigen-antibody complexes were captured with protein A/G Dynabeads (Life Techn. #11202D or #11204D), 5x washed in Fast-IP buffer. Reverse crosslink was performed overnight at 65°C and proteins degraded with 100 uM proteinase K. The DNA was puriefied using Quiagen PCR purification kit according to manufacture?s manual (#28006).
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Library strategy |
ChIP-Seq |
Library source |
genomic |
Library selection |
ChIP |
Instrument model |
Illumina HiSeq 4000 |
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Data processing |
Reads were aligned to the mouse mm10 (GRCm38.6) or Drosophila melanogaster dm6 (BDGP6 release 78) reference genome using BWA-MEM version 0.7.13 with default parameter settings. PCR duplicates were removed using Picard Tools version 2.0.1. Peaks were called using MACS2 version 2.1.1.20160309 in paired-end mode with a FDR threshold of 0.05. Blacklisted regions (http://mitra.stanford.edu/kundaje/akundaje/release/blacklists/mm10-mouse/mm10.blacklist.bed.gz) were removed from the called peaks. Peaks were annotated to the closest TSS using the ChiPpeakAnno R package v3.18.2. (doi: 10.1186/1471-2105-11-237) Genome_build: mm10, dm6 Supplementary_files_format_and_content: The .txt files containing the annotated peak positions called with MACS2. The .bw files contain coverage data for visualization in the genome browser.
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Submission date |
Sep 25, 2019 |
Last update date |
Jan 01, 2021 |
Contact name |
Franziska Greulich |
E-mail(s) |
franziska.greulich@tum.de
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Organization name |
TU München
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Department |
Metabolic Programming
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Lab |
AG Uhlenhaut
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Street address |
Gregor-Mendel-Str. 2
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City |
Freising |
ZIP/Postal code |
85354 |
Country |
Germany |
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Platform ID |
GPL21103 |
Series (1) |
GSE138017 |
H3K4methylation in Setd1a loss-of-function Raw264.7 cells upon LPS or Dex+LPS stimulation |
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Relations |
BioSample |
SAMN12842639 |
SRA |
SRX6902441 |
Supplementary file |
Size |
Download |
File type/resource |
GSM4096698_Raw264_GAR2107_mm10_FDR005_peaks_annotated.txt.gz |
1.3 Mb |
(ftp)(http) |
TXT |
GSM4096698_Raw264_GAR2107_mm10_normTodm.bw |
116.6 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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