Instrument: Illumina HiSeq 2500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: Muscle biopsie was flash frozen on dry ice, and total and RNA was harvested using Trizol reagent Libraries were constructed using the Truseq stranded mRNA sample prep kit (Illumina, San Diego, CA,USA) according to the manufacturer instructions. Briefly, poly-A RNAs were purified using oligo-d(T) magnetic beads from 1µg of total RNA. The poly-A RNAs were fragmented into small pieces using divalent cations under elevated temperature and reverse transcribed using random hexamers, Super Script II (Thermo Fisher Scientific, Carlsbad, CA) and Actinomycin D. During the second strand generation step, dUTP substitued dTTP. This prevents the second strand to be used as a matrix during the final PCR amplification. Double stranded cDNAs were adenylated at their 3' ends before ligation was performed using Illumina's indexed adapters. Ligated cDNAs were amplified following 15 cycles PCR and PCR products were purified using AMPure XP Beads (Beckman Coulter Genomics, Brea, CA, USA). Libraries were validated using a Fragment Analyzer (Agilent Technologies, Santa Clara, CA, USA) and quantified using the KAPA Library quantification kit (Roche, Bâle, CHE).