RNA-seq analysis in the R 6/2 model
Samples of striated tissue from 4- and 6-week-old R6/2 mice and respective controls were obtained in triplicate. Total RNA was purified by tissue homogenization using Trizol reagent (Invitrogen). The total RNA extraction was digested with DNAse I to avoid contamination of the sample with genomic DNA. The RNA concentration was then measured with the Quant-iT  RiboGreen RNA Assay Kit (Life Technologies), and its integrity was determined by the Bioanalyzer 2100 kit (Agilent Technologies, Santa Clara, CA, USA). RNA libraries were prepared with the Illumina TruSeq TM RNA Sample Preparation Kit, following the manufacturer's protocol. The rRNA-depleted RNA was fragmented using divalent cations at elevated temperatures. First-strand cDNA synthesis produced single-strand DNA copies from RNA fragmented by reverse transcription. After second-stranded cDNA synthesis, the double-stranded DNA will be repaired at the 3 'ends. Finally, PCR ligated the universal adapters to the cDNA fragments to produce the final sequencing library. After library validation was carried out with the DNA 1000 chip (on the Agilent Technologies 2100 bioanalyzer), the samples were pooled in equal concentrations into one pool and run on the Illumina HiSeq equipment for 100 end sequencing cycles paired.