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Sample GSM4096700 Query DataSets for GSM4096700
Status Public on Dec 31, 2020
Title GAR1961: Raw264.7_DexLPS_input
Sample type SRA
 
Source name Raw264.7
Organism Mus musculus
Characteristics cell type: Raw264.7
strain: Balb/c
Sex: male
genotype: --
spike-in: Drosophila melanogaster
treatment: 16h 1uM Dex, 3h 100ng/ul LPS
fixation: 10min 1% FA
antibody: none
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).
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.
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).
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina HiSeq 4000
 
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.
 
Submission date Sep 25, 2019
Last update date Dec 31, 2020
Contact name Franziska Greulich
E-mail(s) franziska.greulich@tum.de
Organization name TU München
Department Metabolic Programming
Lab AG Uhlenhaut
Street address Gregor-Mendel-Str. 2
City Freising
ZIP/Postal code 85354
Country Germany
 
Platform ID GPL21103
Series (1)
GSE138017 H3K4methylation in Setd1a loss-of-function Raw264.7 cells upon LPS or Dex+LPS stimulation
Relations
BioSample SAMN12842637
SRA SRX6902443

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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