GSM6797813: human mito post exercise participant 9 [hmt-09-2]; Homo sapiens; miRNA-Seq
Mitochondria were isolated from ~60mg skeletal muscle tissue with the MACS Mitochondria Isolation Kit (human) (cat# 130-094-532, Miltenyi Biotec). Following isolation, the mitochondrial fraction was resuspended in 50 μL ice-cold Storage Buffer (cat# 130-094-532, Miltenyi Biotec) and incubated with 312 μg RNase-A (cat# 19101, Qiagen) for 1 h at 37°C. Following incubation, RNase-A was degraded by the addition of 5 μL proteinase-K (stock concentration 600 mAU/mL) (cat# 19131, Qiagen) and inverting the tube briefly. The solution was centrifuged at 8,000g for 10-minutes at 4C. The supernatant was aspirated, and the mitochondria pellet was washed twice with 100 μL ice-cold Storage Buffer (cat#130-096-946, Miltenyi Biotec), and centrifuged at 13,000g for 2-minutes at 4C between washes. The mitochondria pellet was resuspended in 100 μL storage buffer (cat# 130-094-532, Miltenyi Boitec) and stored at -80°C until further analyses.  For RNA extraction from isolated mitochondria, frozen samples were suspended in 5x volumes of TriReagent (cat# R2061, Integrated Sciences), thawed on ice and then centrifuged at 16,000xg for 2 min at 4°C to pellet the mitochondria. The mitochondria pellet was first sheared 20x through a fine pipette tip. Total RNA was extracted from isolated mitochondrua using Zymo Direct-Zol Micro Prep kit (#R2061, Integrated Sciences) with on-column DNase-I digest (#E1010, Integrated Sciences) as per the manufacturer's protocol. RNA was eluted in 12ul NFW. Small RNA libraries were prepared using the NEBNext Multiplex Small RNA Library Prep Set (cat# #E7560S, New England Biolabs.) with modifications as described by our group previously (Silver and Trewin, 2022, https://doi.org/10.1101/2022.04.27.489477). (3). Briefly, the 3' and 5' adapters and reverse transcription primer were diluted 0.3x, and 20 end-point PCR cycles were conducted. Following PCR amplification, the cDNA library was cleaned using the Monarch PCR & DNA Cleanup Kit (5 μg) (cat# T1030L, New England Biolabs.) using the 7:1 ratio of binding buffer:sample as per the manufacturer's instructions. Uniquely indexed libraries were placed into an equimolar pool. Five aliquots of this pool were run in separate lands of a 6% Novex TBE-polyacrylamide gel (cat# EC6265BOX, Thermo Fisher Scientific), with 5 μL Quick-Load pBR322 DNA-MspI Molecular Marker (cat#E7560S, New England Biolabs.) run in a separate lane. Bands corresponding to the 143 and 190bp molecular markers were manually excised and extracted from the gel as per the manufacturer's protocol (cat#E7560S, New England Biolabs.).