title: HFD_E2_8_Sample27_H3K27ac
organism: Mus musculus
description: Protocols: Mouse livers were perfused and washed with PBS. Livers were then fixed in 1% formaldehyde, the reaction quenced with 2.5M Glycine and kept at -80°C until use. Livers were homogenized using a douncer and washed twice with ice-cold PBS. 5 to 6-week old male mice were fed a control or high-fat diet for 13 weeks. A subset of mice fed a high-fat diet was additionally injected intra-peritoneally with the estrogen receptor agonists 17-beta estradiol (E2) or Diarylproprionitrile (DPN) every other day for the last three weeks of the diet. Control animals underwent sham-injections in the same period. Sample numbers indicate individual mice. Matching RNAseq data for the same mice is available under E-MTAB-11833. Raw sequencing output was demultiplexed and converted to fastq files by the inbuilt Illumina bcl2fastq tool. Individual lanes were contatenated to one fastq file per sample (cat file1.fastq file2.fastq file3.fastq file4.fastq > file5.fastq). Files were trimmed using trimmomatic v0.36. Crosslinked cells were lysed and nuclei extracted as described in detail in Schmidt et al., Methods, 2009. 10.1016/j.ymeth.2009.03.001 Chromatin was sonicated using a Sonics Vibra cell VCX 750 set to 32% duty cycle for 30 cycles (30s on, 59s off).ChIP was performed using antibodies against H3K27ac (Abcam, #4729, lot: GR3211959-1, 5µg) and H3K4me3 (monoclonal, Merck 05-1339, lot: 3305923, 5µg) as previously described in detail in Schmidt et al, Methods, 2009. Sequencing libraries were generated using the Thruplex DNA-Seq Kit (TakaraBio), size-selected and the quality assessed using Bioanalyzer DNA High Sensitivity chips (Agilent Technologies) according to manufacturer's protocols. Libraries were quantified using KAPA quantification kit (Roche).