title: GSM2908155: Ov/Ob Pre; Homo sapiens; MNase-Seq
library_construction_protocol: Skeletal muscle biopsies were obtained from the vastus lateralis muscle using the percutaneous muscle biopsy technique before (Pre) and immediately following (Post) the exercise testing trial. Briefly, 2.5 mL of a 1% lidocaine solution was injected into the thigh and a 6 mm Bergstrom biopsy needle (Pelomi Medical, Denmark) under suction was used collect the muscle sample. The collected sample was quickly rinsed in PBS and blotted to remove any blood prior to freezing. Pre biopsy samples were taken from the participant's non-dominant leg, whereas Post biopsies were obtained from the dominant leg. Samples were then flash frozen in liquid nitrogen and stored at −80°C until further processing. Mononucleosomal DNA was isolated from approximately 10 mg of skeletal muscle (n=5 per group). Briefly, the samples were homogenized using a dounce homogenizer in a 0.25 M sucrose buffer (0.25 M sucrose, 10 mM Tris-acetate pH 8.1, 1 mM EDTA, 1 mM DTT, 1 mM sodium orthovanadate, and 1 1X complete protease inhibitor tablet (Roche, Indianapolis, USA). The pellets were washed and resuspended in 0.25 sucrose buffer containing 0.1 mM CaCl2 and 4 mM MgCl2 and incubated with micrococcal nuclease (MNase; Roche, Indianapolis, USA) for isolation of mononucleosomal DNA for 15 min at 37°C. Then, EDTA was added (final concentration: 5 mM). After centrifugation, the resulting pellets were suspended in with 0.25 M lysis buffer (50 mM Tris, pH 8.1, 10 mM EDTA, 1% SDS, and 1 1X complete protease inhibitor tablet (Roche, Indianapolis, USA). Samples were treated with 0.1 mg/mL of proteinase K (Qiagen, Germantown, MD) overnight at 37°C. Mononucleosomal DNA sequencing was performed by the Genomics Core Facility at Pennington Biomedical Research Center. The NEB ultra II DNA library prep kit and NEBNext multiplex oligos for Illumina index primer sets 1 and 2 (New England Biolabs, Ipswich, MA) were used for library construction, per the manufacturer's protocol. Sequencing was completed using the NextSeq 500 Sequencing System (Illumina, San Diego, CA) to generate 75 nucleotides long single-end reads, with an average of ~387 million reads per treatment group.