Qiazol lysis

Total RNA extraction from myotubes was performed using QIAzol Lysis reagent and RNeasy Mini Kit with on‐column DNase digestion (QIAGEN). RNA concentration was determined with NanoDrop ND2000 spectrophotometer (Thermo Scientific). Total RNA was reverse transcribed using SuperscriptTM III First‐Strand Synthesis SuperMix for qRT‐PCR (Invitrogen). The relative mRNA levels were determined by real‐time PCR using Taqman method in a 96‐well format on a Mastercycler ep Realplex (Eppendorf) for mouse muscle atrophy F‐box protein 32 (Atrogin1/Fbxo32), Cathepsin‐L (Ctsl), Insulin‐like growth factor‐1 (Igf1), Muscle‐specific RING finger protein 1 (MuRF1/Trim63) and Peroxisome proliferator‐activated receptor gamma coactivator 1‐alpha (Pgc1), using the following Taqman probes: Mm499523_m1, Mm00515597_m1, Mm00439560_m1, Mm01185221_m1, and Mm01208835_m1, respectively. After testing a number of different potential candidates Mouse 18S (hs99999901_s1), Cyclophilin (Mm02342429_g1), and RPLPO (Mm01974474_gH) were adopted as reference genes. Triplicates of each of three experimental replicates for each experimental condition were performed. Amplification efficiencies for each probe were empirically determined and relative quantification was calculated using the Pfaffl method adjusted to normalize for all three reference genes (Pfaffl et al. 2002).

After cutting skin samples into small pieces, samples were homogenized and lysed by using Tissue Raptor II and QIAzol^® Lysis protocol (QIAzol^® Handbook 2021, QIAgen, Crawley, UK). The quality of RNA was routinely assessed by determining the A260:A280 ratio using NanoDrop2000 (Thermo Scientific, UK). RNA libraries were created using TruSeq stranded Total RNA with ribozero GOLD (illumina, UK) and sequenced on an illumina HiSeq 2500 platform with 2 x 75 bp reads to the depth of 13.5-22.8 million reads per sample.