GSM4823200: 17; Homo sapiens; RNA-Seq
Subjects were fasted and refrained from exercise and alcohol 24 hours prior to testing. Biopsies from vastus lateralis were collected using the suction modified Bergström technique from the non-dominant leg under local anaesthesia using a using a 5-mm Bergström o needle. Collected tissue was snap frozen in liquid nitrogen and stored at -80°C until further analysis. Total RNA was extracted by grinding frozen muscle tissue and dissolving approximately 30 mg in 1 mL Trizol reagent in Tissue lyser II (Qiagen) for 2 min at 30 Hz. 240 µL of cold chloroform was added to the lysates, shaken thoroughly for 30 seconds and incubated on ice for 5 min before centrifugation at 12,000 RPM for 15 min at 4˚C (Mikro 200R, Hettich, Germany). The aqueous layer was transferred to a new tube and 700 µL of ice-cold isopropanol was added, mixed with RLT buffer (Qiagen) and loaded onto RNAeasy spin columns (Qiagen). RNA purification was done according to manufacturers' protocol. RNA concentration was measured using NanoDrop spectrophotometer (Thermofisher). RNA integrity was measured using RNA Screentape on the 2200 Tapestation (Agilent). RNA preparation, library construction and sequencing on BGISEQ-500 was performed at Beijing Genomics Institute.