TempO-seq of Rattus norvegicus: kidney
Total RNA was extracted from liver and kidney using the RNeasy Mini Kit (Qiagen) with a DNA digestion step. RNA purity and quality were determined by the absorbance ratio (260/280) and calculation of the RNA integrity number, respectively. RNA was sent frozen to BioSpyder (Carlsbad, California) for HTT analysis using the rat S1500+ TempO-Seq platform (Mav et al., 2018, https://ntp.niehs.nih.gov/results/tox21/s1500-gene-set-consensus-strategy-index.html; last accessed June 17, 2020). The S1500+ measures approximately 3000 transcripts covering nearly 90% of all well-curated gene sets. Briefly, mRNA targets were hybridized with a detector oligo (DO) mix (2 DOs per transcript) in 96-well plates, followed by nuclease digestion of excess oligos, ligation, and elution to generate a pool of amplification templates that share common PCR primer-binding sites. During product amplification, each sample well was assigned a specific, barcoded primer pair, allowing for proper identification and matching of mRNAs and samples following sequencing. Sample amplicons were pooled and cleaned up using a PCR clean-up kit. Libraries were then sequenced using a HiSeq 2500 Ultra-High-Throughput Sequencing System (Illumina, San Diego, California). Sequencing readouts were demultiplexed to generate FASTQ files.