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| Status |
Public on Dec 31, 2020 |
| Title |
Raw264.7_wt_LPS_aH3K4me1_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: wt
spike-in: Drosophila melanogaster
treatment: 16h 0.1% EtOH, 3h 100ng/ul LPS
fixation: 10min 1% FA
antibody: rabbit polyclonal a-H3K4me1 Diagenode #C15410194
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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).
Library preperation was performed from 2 ng of ChIP DNA using the Kapa Hyper Prep Kit with PCR library amplification (#KK8504, KapaBiosystems) according to the manufactur's protocol. Shortly, ChIP DNA was end-repaired and A-tailed before adapter ligation. After adapter ligation, the ChIP DNA was purified with Agencourt AMPure XP beads (#A63880, Beckman Coulter) and size-selected for 360-600 bp using the Pippin Prep System from Saga Science. The size-selected library is amplified by PCR using the Kapa Hyper Prep Kit (#KK8504,KapaBiosystems) and purified terminally with Agencourt AMPure XP beads (#A63880, Beckman Coulter).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
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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 |
Dec 05, 2020 |
| 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 |
GPL24247 |
| Series (1) |
| GSE138017 |
H3K4methylation in Setd1a loss-of-function Raw264.7 cells upon LPS or Dex+LPS stimulation |
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| Relations |
| BioSample |
SAMN17006373 |
| SRA |
SRX9635400 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4957878_Raw264_wt_3h100ngLPS_rep3_aH3K4me1_MUC21198_mm10_FDR005_peaks_annotated.tab.gz |
1.4 Mb |
(ftp)(http) |
TAB |
| GSM4957878_Raw264_wt_3h100ngLPS_rep3_aH3K4me1_MUC21198_mm10_normTodm.bw |
457.8 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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