GSM5237466: Skeletal Muscle_Rat5; Rattus norvegicus; ATAC-seq
Tissue samples were transferred to 1.1 ml micronic tubes (Micronic, USA) and resuspended in 600 µl of homogenization buffer (320 mM Sucrose, 10 mM Tris-HCl pH 7.8, 100 nM EDTA, 5 mM CaCl2, 3 mM Mg(AC)2, 167 µM β-mercaptoethanol, 1X protease inhibitor). Three, 2.8 mm ceramic beads (Omni International, Kennesaw, GA, USA) were added to each micronic tube. Tubes were vortexed briefly and kept on ice. The tissue samples were homogenized in a Bead Ruptor Elite bead mill homogenizer with Omni BR Cryo cooling unit (Omni International) using 2 cycles of the following settings: speed: 1.0, time: 20 sec and speed: 2.1, time: 20 sec, dwell time: 20 sec at both speeds, while cooled by liquid nitrogen to 10°C. Following homogenization, each tissue homogenate was filtered through a mini 20 µm pluriStrainer (pluriSelect, Leipzig, Germany). The number of nuclei in the filtrate was counted using an Automated Cell Counter (Cellometer K2 Image cytometer, Nexcelom) following staining with the Propidium Iodide (PI) dye. Approximately 75,000 nuclei were transferred into a fresh tube and diluted in 1 ml of ATAC-seq resuspension buffer [ATAC-seq RSB (Corces et al., 2017) + 0.1% Tween 20 (Sigma, St. Louis, MO, USA)]. Nuclei were centrifuged using Centrifuge 5418 R (Eppendorf) at 725 RCF for 10 minutes, and the supernatant was carefully aspirated. Nuclei were incubated in 50 µl transposition reaction mixture containing 25 µl of 2X TD buffer (Illumina, Inc., San Diego, CA, United States), 16.6 µl of PBS, 0.5 µl of 1% digitonin (Sigma), 0.5 µl of 10% Tween-20, 1.5 µl of Tn5 transposase (Illumina, 0.8 U/μl), and 6 µl of nuclease-free water using a thermomixer (USA Scientific, Ocala, FL, USA) set at 37 °C for 30 min with a speed of 1000 rpm. Transposed DNA was purified using a DNA clean and concentrator kit (Zymo Research, Irvine, CA, USA), according to the manufacturer's protocol. Following PCR amplification, libraries were purified using Agencourt Ampure XP beads (Beckman Coulter, Indianapolis, IN, USA) double-size selection (0.5x:1.3x). Library quality was assessed using the Agilent Bioanalyzer High-Sensitivity DNA kit and the Qubit dsDNA high sensitivity assay kit (Thermo Fisher). All libraries were pooled and sequenced with 100-bp paired-end reads on the Illumina Novaseq 6000 at the New York Genome Center.