ATAC-Seq Invivo Control HFD Diet

Liver tissue was homogenized in PBS via mortar and pestle on ice. Samples were centrifuged at 300 rpm for 5 minutes at 4°C. Cells were resuspended in 1 ml ATAC lysis buffer provided in the ATAC-Seq kit (Active Motif, 105310). Nuclei were counted and 5x104 nuclei per reaction were used. ATAC-Seq was performed using the ATAC-Seq kit (Active Motif, 105310) following the protocol provided by the manufacturer. Therefore, the tagmentation reaction was performed by adding 50 µl of Tagmentation Master Mix to each sample. The reaction was incubated at 37°C for 30 minutes in a Thermoshaker (Life Technologies). Samples were transferred to new tubes and 250 µl DNA Purification Binding Buffer and 5 µl 3 M sodium acetate was added to each sample. No pH adjustments had to be performed. DNA purification was performed using DNA purification columns provided by the kit. The purified DNA was quantified using the Qubit instrument (Thermo Scientific). Sequencing libraries were prepared using NEBNext Ultra library preparation kit for PCR amplification following the manufacturer’s instructions. DNA clean-up was performed using 1.2x the sample volume of SPRI beads provided by the kit. The purified DNA was assessed for quality and quantity via the BioAnalyzer (Agilent). Generated libraries were sequenced on an Illumina NovaSeq instrument with an average depth of 50 million reads per library (150 bp, paired-end).