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| Status |
Public on Jul 23, 2021 |
| Title |
20L004205: Het_H3_ChIP_Rep2 |
| Sample type |
SRA |
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| Source name |
Visceral white adipocyte tissue
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
genotype: Mof heterozygous
tissue: Visceral white adipocyte
chip antibody: H3 (milipore, #06-755)
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| Treatment protocol |
Standart Diet
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
WAT of 8 week old SD treated animals was minced and transferred into a Dounce homogenizer (loose pestle), covered with 1% formaldehyde in PBS, and dissociated, followed by 15 min incubation at room temperature (RT). During incubation, the tissue suspension was filtered using a nylon 70 µM cell strainer (Falcon, 352350). 125 mM glycine final was added to the sample, incubated at RT for 5 min and the suspension was pelleted for 5 min at 500 g. Cell pellets were washed twice in ice cold PBS and pelleted for 5 min at 500 g. For NEXON based nuclei isolation cell pellets were resuspended in 1ml of lysis buffer (10mM Tris-HCl pH 8, 10mM NaCl, 0.2% Igepal, protease inhibitor cocktail. Cell suspension was transferred into Covaris MilliTube (cat. No. 520130) and sonicated in Covaris instrument (E220) for 30 sec at peak power 75W, duty factor 10% and 200 cycles/burst. Nuclei were pelleted at 1000g at 20°C for 5min. Pellets were resuspended in 0.5% SDS and incubated at RT for 10min. Sample was diluted 1:5 in the digestion buffer (1.1% Triton, 1x CutSmart buffer , H2Odest). Up to 1*10^6 nuclei were digested with Cvik1 (5U/100.000 cells) at 25°C for 17hrs, shaking at 800rpm. Nuclei were pelleted for 5min at 1000g and washed in a nuclei wash solution (10mM Tris-HCl pH 8, 0.25% Triton, 0.2mg/ml BSA ) until residual fat was removed. Nuclei were pelleted at 5000g for 10min, resuspend in the shearing buffer (10mM Tris-HCl pH 8, 0.1% SDS, 1mM EDTA), transferred into Covaris MicroTube (cat. No. 520052) and sonicated for 6min at peak power 105W, duty factor 2% and 200 cycles/burst. Sample was diluted 1:6 in 1x iC1 buffer (iDeal ChIP-seq, Diagenode) and incubated with 2ug of antibody o/n at 4°C. The next day protein G Dynabeads were added and incubated for 3 hrs at 4°C. Beads were washed and DNA was eluted according to manufacturer's instructions (iDeal ChIP-seq, Diagenode). DNA was purified using MinElute PCR purification kit (Qiagen) for further analysis
Libraries were prepared with the NEBNext Ultra DNA Library Prep Kit for Illumina according to the manufacturer’s instructions (New England Biolabs, #E7645). ChIP libraries were sequenced using an Illumina NovaSeq6000 sequencer with 2 × 100 base paired-end reads.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
8 weeks animals under normal diet
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| Data processing |
ChIP-seq datasets were mapped to GRCm38/Mm10 with default paired-end parameters from snakePipes version 2.1.2 (Bhardwaj et al. 2019). Peaks were called with MACS2 (Galaxy version 2.1.1.20160309.3), and bandwidth was set to 200, lower mfold bound to 5, upper mfold bound to 500, and the q-value to 0.1. Input was used as control to call peaks. Peaks from each cell type were merged using cat, BEDtools sort (Galaxy version 2.27.0.0) and piped to BEDtools merge with a distance of 1000 bp. BamCompare (Galaxy version 2.5.1.0.0) with a bin size of 50 bp. The data were normalized as log2 fold change over H3 immunoprecipitation. For plotting heatmaps were generated using deepTools2 compute matrix and plotHeatmap function from DeepTools2 version 3.5.0 (Ramírez et al. 2016). Box-plots were generated using DeepTools2 multiBigwigSummary scores. The Bioconductor package ChIP-Seeker (Yu et al. 2015). was used to retrieve the nearest genes around the peak, annotate the genomic region of the peak, and peak features annotation. GO term analysis was performed using the Metascape (Zhou et al. 2019) platform and the ShinyGo v0.61 database (Ge et al. 2020).
Genome_build: mm10
Supplementary_files_format_and_content: bigwig files having log2-fold change scores
Supplementary_files_format_and_content: peak bedfiles
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| Submission date |
Aug 19, 2020 |
| Last update date |
Jul 23, 2021 |
| Contact name |
Asifa Akhtar |
| E-mail(s) |
akhtarlab_data@ie-freiburg.mpg.de
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| Organization name |
Max Planck Institute of Immunobiology and Epigenetics
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| Department |
Chromatin Regulation
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| Lab |
Akhtar Lab
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| Street address |
Stuebeweg 51
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| City |
Freiburg |
| ZIP/Postal code |
79108 |
| Country |
Germany |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE156462 |
MOF haploinsufficiency triggers diet-induced obesity resistance (ChIP-seq) |
| GSE156463 |
MOF haploinsufficiency triggers diet-induced obesity resistance |
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| Relations |
| BioSample |
SAMN15856735 |
| SRA |
SRX8969360 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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