Modulating Molecular Chaperones Improves Mitochondrial Bioenergetics and Decreases the Inflammatory Transcriptome in Diabetic Sensory Neurons
100 ng of total RNA was used to prepare cDNA libraries with the TruSeq Stranded Total RNA Library Prep Kit (Illumina Inc., San Diego, CA). In general, poly(A)+ containing mRNA was purified with oligo-dT attached magnetic beads. Purified mRNA was then fragmented and primed with random hexamers, which were subsequently reverse transcribed into first strand cDNA using reverse transcriptase. The RNA templates were then removed from the single-strand cDNA and a replacement strand was synthesized to generate double-strand cDNA. The double-strand cDNA was end-repaired and adenylated at the 3’ ends. For each sample, Illumina-specific adaptors that contain a unique 6 bp index sequence that identifies each sample were ligated to the 3’ adenine-tail of the fragments to permit the single read, multiplex sequencing of transcripts within each lane of the flow cell. cDNA products (~260 bp) were amplified and submitted to quality control with the Agilent 2200 Tape Station. cDNA libraries prepared from the 3-5 biological replicates from each treatment group were then randomly assigned to two lanes of the flow cell for subsequent single-end sequencing with a read length of 100 bases using the Illumina HiSeq 2500 System (Illumina Inc., San Diego, CA).