GSM8524367: human skm 3hpost exercise participant 5 (miRNA-seq); Homo sapiens; miRNA-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. Small RNA libraries were also prepared from 299 ± 167 ng (n=35) total RNA extracted from whole skeletal muscle tissue using the NEBNext Multiplex Small RNA Library Prep Set (cat# #E7560S, New England Biolabs.) with 3' and 5' adapters and reverse transcription primer diluted 0.5x, and 15 PCR cycles to amplify the adapter-ligated library. The cDNA was cleaned, quantified and assessed as described above for mitochondrial small RNA libraries.