Truseq stranded mrna library prep protocol

Total RNA was extracted in triplicate using the RiboPure RNA Purification Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. Transcriptome libraries were constructed using the TruSeq Stranded mRNA Library Prep protocol (Illumina) according to the manufacturer’s instructions. Total RNA was used for library preparation, which was checked for quality using the Agilent 2100 Bioanalyzer (Santa Clara, USA). Library quantitation was performed by Qubit using the Qubit RNA BR Assay Kit (Thermo Fisher Scientific). Libraries were normalized, pooled, and sequenced using the Illumina MiSeq sequence machine. For sequencing, the 150 bp single-read sequencing chemistry was performed using the MiSeq Reagent Kit v3 (Illumina, San Diego, California, USA) according the manufacturer’s protocol. Raw reads were exported and analyzed in CLC Genomics Workbench (version 12, Qiagen Aarhus A/S, Denmark), trimmed and mapped against ncRNAs to discard rRNAs and tRNAs. Expression values were reported as RPKM (Reads Per Kilobase of transcript per Million mapped reads) [34 (link)]. Statistical analyses were performed using the DESeq2 package [35 (link)] in R Studio.

Bulk RNAseq was performed on frozen muscle biopsy specimens as previously described.^{6 (link),9 (link)–13 (link)} Briefly, RNA was extracted with TRIzol. Libraries were either prepared with the NeoPrep system according to the TruSeq Stranded mRNA Library Prep protocol (Illumina, San Diego, CA) or with the NEBNext Poly(A) mRNA Magnetic Isolation Module and Ultra^™ II Directional RNA Library Prep Kit for Illumina (New England BioLabs, ref. #E7490, and #E7760).