GSM8508306: human skm 3hpost exercise participant 9; Homo sapiens; RNA-Seq
For RNA extraction from skeletal muscle tissue, 10-15 mg frozen human skeletal muscle tissue was submerged in 400 μL ice-cold 1x DNA/RNA protection reagent (T2010, New England Biolabs, MA, USA) containing 700-800 mg 1.0mm zirconia/silica beads (11079110z, BioSpec Products, St. Helena, Australia). The tissue was homogenised with mechanical disruption at 6,500rpm for 30 s (MagnaLyser, Roche Diagnostics, NSW, Australia). Next, 30 μL Prot K Reaction Buffer (T2010, New England Biolabs, MA, USA) plus 15 μL Prot K (T2010, New England Biolabs, MA, USA) was added to the tissue homogenate and incubated at 55°C for 5 min. Following incubation, the lysate was briefly vortexed and then centrifuged at 16,000xg for 2 min. The supernatant was transferred to a new tube. RNA extraction was then performed using the Monarch Total RNA Mini Prep Kit (T2010, New England Biolabs, MA, USA) with on-column DNase-I digest as per the manufacturer's instructions. RNA was eluted from the column in 30 μL nuclease-free water. The eluted RNA was tested for purity and concentration on a Nanodrop One (Thermo Fisher Scientific, MA, USA). If required, RNA was diluted below 25 ng/μL and then the RNA integrity number (RIN) was determined using the HS RNA screentape and reagents (5067-5579 and 5067-5580, Agilent Technologies, CA, USA) on the Agilent 4200 TapeStation. Total RNA extracted from whole skeletal muscle tissue was prepared for total RNA sequencing externally (Macrogen, South Korea). Total RNA libraries depleted of ribosomal RNA were prepared using the TruSeq stranded Total RNA Library Preparation with RiboZero (human/mouse/rat).