GSM4008589: Ov/Ob Pre 15; Homo sapiens; RNA-Seq
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. Skeletal muscle samples (n=14 per group for lean Pre and lean Post and n=15 per group for Ov/Ob Pre and Ov/Ob Post) were homogenized (Polytron homogenizer, Kinematica AG, Lucerne, Switzerland) in Trizol reagent (Thermo Fisher Scientific, Waltham, MA) per the manufacturer's protocol. Following phase separation, the aqueous layer was removed and column purified using the RNeasy kit (Qiagen, Germantown, MD). RNA-seq sample preparation and sequencing was performed by the Genomics Core Facility at Purdue University. cDNA libraries were constructed using the TruSeq stranded mRNA library prep kit (Illumina, San Diego, CA), per the manufacturer's protocol and were subjected to sequencing on the Illumina HiSeq 2500 Ultra-High-Throughput Sequencing System (Illumina, San Diego, CA). 100 nucleotides long single-end reads were generated, with an average of ~42 million reads per treatment group.