title: E2_HFD_male_4
organism: Mus musculus
description: Protocols: Mouse livers were perfused and washed with PBS and flash-frozen in liquid nitrogen immediately after animal sacrifice and stored at -80C until use. 5 to 6-week-old male and female C57BL/6J mice obtained from in-house breeding were put on a control (D12450J, 10% kcal fat, Research Diet) or high-fat diet (D12492, 60% kcal fat, Research Diet) for 13 weeks. Subsets of male and female mice on HFD were additionally injected interperitoneally with the estrogenic ligands 17-beta-estradiol (E2, 0.5 mg/kg body weight, Sigma Aldrich), 4,4',4''-(4-Propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT, 2.5 mg/kg body weight, Tocris), 2,3-Bis(4-hydroxyphenyl)propionitrile (DPN, 5 mg/kg body weight, Tocris) and 4-(2-(3,5-dimethylisoxazol-4-yl)-1H-indol-3-yl)phenol (DIP) or given a sham injection every second day from week ten to week 13. The ligands were diluted in 55 % water, 40 % PEG400 and 5% DMSO. 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). Approximately 20 µg of flash-frozen liver tissue was homogenized in 700 µL Qiazol (Qiagen) using a TissueLyzer II (Qiagen, 2min, 25 Hz, 2 times). The samples were incubated on room temperature for 5min, before adding 140 µL chloroform (Sigma Aldrich). This mixture was shaken for 15s, incubated for 3min, and centrifuged at 9000 x g and 4°C for 5min. The aqueous phase was carefully removed, and an equal volume of isopropanol added. This mixture was incubated at room temperature for 10min, before centrifugation at 20.000 x g and 4°C for 10min. Supernatant was removed and the pellet washed twice with 70% ethanol, air-dried and resuspended in water. The isolated RNA was DNase treated using the Turbo DNase Kit (ThermoFisher) according to the manufacturer's instructions. In brief, 10 µg of RNA was treated with 2U DNase and 40U RNaseOUT (Thermo Fisher) at 37°C for 30min, before treatment with DNase inactivation reagent for 5min under constant homogenization. The sample was centrifuged at 10.000 x g for 2min to remove the inactivation reagent. To purify the obtained DNase-treated RNA, the RNA was diluted to 130 µL with water, before adding 20 µL sodium acetate (Thermo Fisher, 3M, pH 5.2), 1 µL GlycoBlue (ThermoFisher) and 600 µL ice-cold 99.8% ethanol. Next, the RNA was precipitated at -80°C overnight, before centrifugation at 20.000 x g for 30min, washing the pellet twice with 70% ethanol, air-drying and resuspending in water. The RNA quality was assessed on a Bioanalyzer 2100 device using RNA Nano chips (Agilent) and only high quality RNAs (RIN > 6.5) were used for RNA-sequencing. Strand-specific RNA libraries were generated using the NEBNext Ultra II stranded library kit (NEB) combined with polyA-coupled beads (NEB) according to the manufacturer's instructions. The library quality was assessed on a Bioanalyzer 2100 device using DNA High Sensitivity chips (Agilent) and quantified using a KAPA library quantification KIT (Roche).