GSM9008819: Mouse liver, atorvastatin, rep3; Mus musculus; ATAC-seq
Nuclei Isolation from Mouse Liver and ATAC-Seq Library Preparation Liver pieces with a weight range of 100–160 mg (average ~120 mg) were used. Liver pieces were frozen in liquid nitrogen and stored at -80°C in 2-mL tubes. Liver samples were briefly thawed from -80°C, kept on liquid nitrogen, and cut into small (rice grain-sized) pieces using tweezers and a scalpel. Liver pieces were homogenized in five hundred microliters of chilled 0.1X Lysis Buffer with an RNase-Free Disposable Pellet Pestle. The homogenized mixture was pipetted through the cut tips 10–15 times with a 1-mL tip. Samples were incubated on ice for 10 minutes. Five hundred microliters of chilled Wash Buffer were added with a normal pipette tip and mixed by pipetting 5–10 times. The tube was spun briefly to shift the non-homogenized debris to the bottom. The sample was passed through a 70 μm Flowmi Cell Strainer into a new 2-mL tube. A maximum of 300 μL was transferred per strainer. Non-homogenized debris was not transferred. The sample was then passed through a 40 μm Flowmi Cell Strainer into a new 2-mL tube, transferring a maximum of 300 μL per strainer. Samples were centrifuged at 500 g for 5 minutes at 4°C. The supernatant was discarded, and the nuclei pellet was resuspended in 1 mL ATAC-RSB. Samples were centrifuged again at 500 g for 5 minutes at 4°C. The supernatant was discarded, and the nuclei pellet was resuspended in 600 μL ATAC-RSB. The sample was passed through a 40 μm Flowmi Cell Strainer into a new 2-mL tube. ATAC-RSB was added to achieve a final volume of 1 mL. Nuclei isolation was verified with Trypan Blue staining. Nuclei were counted, and 100,000 nuclei were taken for tagmentation. For subsequent ATAC-Seq analysis, 100,000 nuclei were subjected to a Tn5 transposition reaction. This was carried out using 2.5 µL of TDE1 enzyme from the Nextera DNA Library Prep Kit (Illumina, FC-121-1030). The transposition reaction mix was incubated for 45 minutes at 37°C with shaking at 800 rpm. DNA was then purified using the MinElute PCR Purification Kit (Qiagen, 28004). The transposed DNA was PCR-amplified and barcoded using previously published primers (REF) along with the NEBNext High-Fidelity 2× PCR Master Mix (New England Biolabs, M0551S). The optimal number of PCR cycles was determined as described previously (REF). After amplification, the DNA was size-selected with SPRIselect beads (Beckman Coulter, #B23317) to eliminate fragments smaller than 150 bp and larger than 800 bp, following the manufacturer's protocol. The quality of size selection, library concentration, and fragment size distribution was assessed using the Agilent Bioanalyzer High Sensitivity DNA Analysis Kit (Agilent, 5067-4626). Two biological replicate samples were sequenced using the Illumina NextSeq 500 platform (paired-end 40 bp reads).