MinION sequencing
Muscles from anterior thigh were isolated, finely minced using surgical scissors, and resuspended in 2 ml of serum-free RPMI supplemented with 1000U/ml Collagenase from Clostridium histolyticum (Sigma-Aldrich, C9407), 250 μg/ml DNAse I (Sigma-Aldrich, 10104159001) and 8 μg/ml Dispase I (Sigma-Aldrich, D4693). Samples were incubated for 30 min in 37°C with 140 rpm agitation. Digested muscle suspensions were put on ice and processed in 4°C from now on. 10 ml of ice cold FACS buffer (0.2% BSA/PBS) was added to muscle suspensions, following straining with a 70 μm nylon strainer (Greiner, 542070). With strainer on, the suspension was centrifuged at 300×g for 7 min, crude pellet was subjected twice to ACK (150 mM Ammonium chloride, 1mM Potassium bicarbonate, 0,1 mM Disodium EDTA, pH = 7.2) treatment for 2 minutes on ice, following washing with 10 ml FACS buffer, each time centrifugation was set to 300×g for 7min. Crude pelet was resuspended in 1ml FACS and strained through 30 um cap strainer of FACS tube. With caps on, the samples were centrifuged at 800×g for 3 min in 4°C and supernatant discarded, following resuspension in 2 ml FACS buffer. Equal volumes of samples were mixed thoroughly with 33% Percoll (# GE17-0891-01) in PBS and spun in 4°C at 800×g for 30 min. RNA from cells was isolated using ArcturusTM PicoPureTM RNA Isolation Kit (AppliedBiosystemsTM, KIT0204) following guidelines for RNA isolation from Cell Pellets with on-column DNAse treatment (Qiagen, 79256). To assess the integrity of isolates, each RNA sample after DNAse treatment was analyzed with Agilent 2200 TapeStation system, using Agilent High Senitivity RNA ScreenTape (Agilent, 5067-5579). Direct RNA sequencing was performed as described previously (Bilska et al., 2020). 3.5-5 μg of total mRNA was mixed with 50-200 ng oligo-(dT)25-enriched mRNA from Saccharomyces cerevisiae yeast to optimize sequencing efficiency, and standards with predefined poly(A) lengths, and processed with a Direct RNA Sequencing Kit (catalog no. SQK-RNA002, Oxford Nanopore Technologies) according to the manufacturer's instructions . Sequencing was performed with MinION device (Oxford Nanopore Technologies, Flow cell type FLO-MIN106; RevD).