GSM9329385: mutant day 2 rep3; Mus musculus; RNA-Seq
Tibialis muscles were homogenized in TRIzol reagent (Life Technologies, Darmstadt, Germany). RNA was isolated using a standard phenol/chloroform extraction protocol, and quantified by spectrophotometry (Nanodrop, Thermo Fisher). P3129 : RNA-Seq libraries were generated from 300 ng of total RNA using Illumina Stranded mRNA Prep, Ligation kit and IDT for Illumina RNA UD Indexes Ligation (Illumina, San Diego, USA) according to manufacturer's instructions. Following polyA selection, mRNAs were fragmented at 94oC for 8 minutes. DNA library were amplified using 12 cycles of PCR. Surplus PCR primers were further removed by two successive purifications using SPRIselect beads (Beckman-Coulter, Villepinte, France).  The final libraries were checked for quality and quantified using Bioanalyzer 2100 system (Agilent technologies, Les Ulis, France). P2748 : RNA-Seq libraries were generated according to manufacturer's instructions from 500 ng of total RNA using the Illumina Stranded mRNA Prep, Ligation kit and IDT for Illumina RNA UD Indexes Ligation (Illumina, San Diego, USA). Briefly, Oligo(dT) magnetic beads were used to purify and capture the mRNA molecules containing polyA tails.The purified mRNA were then fragmented at 94oC for 8 min and copied into first strand complementary DNA (cDNA) using reverse transcriptase and random primers. Second strand cDNA synthesis further generated blunt-ended double-stranded cDNA and incorporated dTTP in place of dUTP to achieve strand specificity by quenching the second strand during amplification. Following A-tailing of DNA fragments and ligation of pre-index anchors, PCR amplification was used to add indexes and primer sequences and to enrich DNA libraries (30 sec at 98oC; [10 sec at 98oC, 30 sec at 60oC, 30 sec at 72oC] x 11 cycles; 5 min at 72oC). Surplus PCR primers were further removed by purification using SPRIselect beads (Beckman-Coulter, Villepinte, France) and the final libraries were checked for quality and quantified using capillary electrophoresis.