title: NextSeq 550 sequencing; RNAseq-based Transcriptional Profiling of Adult Male Rat Liver and/or Kidney following 90 Day Dietary Exposure to Fenpicoxamid, Pronamide, Sulfoxaflor, or Triclopyr Acid
design: RNAseq-based Transcriptional Profiling of Adult Male Rat Liver and/or Kidney following 90 Day Dietary Exposure to Fenpicoxamid, Pronamide, Sulfoxaflor, or Triclopyr Acid
library_construction_protocol: Liver (upper third of the left lateral lobe) and kidney (transverse section through the middle of the organ) were placed in 10 volumes of RNAlater and stored at 4C for 12-72 hours.  Thereafter, excess RNAlater was removed and the samples stored at <-60C until total RNA extraction. The upper third of the left lateral liver lobe and a transverse section through the middle of the kidney were dissected and placed into RNAlater.  For total RNA isolation, a 30 mg section of the dissected tissue was used. Dietary administration of each compound began on test day 1, when the animals were 6-7 weeks old.  For the Triclopyr Acid study, dietary concentrations of the compound were adjusted weekly throughout the study to maintain a constant dose level in terms of mg/kg/day.  For Sulfoxaflor, Pronamide, and Inatreq studies, dietary concentrations of the compound were kept constant throughout the exposure period.  All animals on all studies were fasted beginning on the afternoon prior to necropsy. Total RNA was extracted from liver and kidney tissue using a Geno/Grinder 2010 homogenizer (OPS Diagnostics, Lebanon, New Jersey, U.S.A.) and a Qiagen RNeasy Mini kit (Qiagen, Venlo, Netherlands) following the manufacturer's protocol.   The Illumina Truseq Stranded mRNA Sample Preparation kit (Illumina, San Diego, California, U.S.A.) was used to generate RNAseq libraries following the manufacturer's recommended protocol.  First, poly-A containing mRNA was purified from 1 µg of total RNA using poly-T oligo-attached magnetic beads.  Purified mRNA was fragmented to approximately 200 bp average length using divalent cations under high temperature.  Following fragmentation, SuperScript II reverse transcriptase and random hexamers were used to synthesize first strand cDNA.  Second strand synthesis was completed using dUTP-containing Second Strand Marking Master Mix.  The resulting blunt end ds cDNA fragments underwent end repair, A-tailing, and ligation to dual-indexed Illumina adapters.  Finally, adapter-ligated library products were enriched with 13 cycles of PCR and purified using AMPureXP beads.  The purified enriched libraries were normalized to 4 nM concentration, pooled, denatured with sodium hydroxide, and diluted in hybridization buffer.  The normalized and pooled library was further diluted to 2 pM prior to sequencing.