GSM8919402: GRC355_CC6_S13; Danio rerio; RNA-Seq
Samples from experiment 1 underwent RNA isolation by Q2 Solutions Expression Analysis, LLC (Morrisville, NC, USA). Samples from experiment 2 underwent RNA extraction at US EPA (Durham, NC, USA) using Direct-zol™ RNA MiniPrep Plus kit (Zymo Research) per manufacturer instructions. RNA sample quality and concentration from both experiments were checked by Bioanalyzer and Nanodrop or Qubit. For Experiment 1, paired-end (50 base pair) total RNA-sequencing libraries were prepared using Illumina TruSeq Stranded Total RNA with Ribo-Zero Gold Library Prep Kit for rRNA depletion (Illumina, San Diego, California, cat. No. RS-1222303) at Q2 solutions Expression Anaysis according to manufacturer directions. Briefly, rRNA was selectively filtered from total RNA samples through streptavidin bead binding and cleanup. Purified RNA then underwent heat fragmentation with divalent cations prior to stranded cDNA synthesis and barcoding of samples using the TruSeq stranded Total RNA kit. cDNA libraries were quantified and cDNA insert size assessed by Agilent Bioanalyzer (DNA 1000 kit #5067-1504) and qPCR (KAPA Library Quant Kit #KK4824, KAPA Biosystems, Wilmington, MA). Sample libraries were then normalized, randomly pooled, and sequenced at 8 samples per lane to a target read depth of >20 million reads on an Illumina HiSeq 2500. For Experiment 2, purified RNA from each sample (500 ng per sample) were randomized and processed on Apollo324 for polyA selection and then continued on library prep with PrepX mRNA 48 Protocol v19 at US EPA (Durham, NC, USA). PCR amplification with 32 index primers were run for 13 cycles. The resulting PCR products were cleaned up on Apollo324 with PCR Cleanup 48 Protocol. The volume of purified libraries was about 10 μl. The PCR product's quality was checked on Bioanalyzer with High Sensitivity DNA chips and the average size (bp) was obtained according to the electropherogram. The collected libraries were quantified by Qubit DNA HS kit. The molar concentration of each library was estimated by using average molecular size from Bioanalyzer data and concentration from Qubit measurement. Then an equal amount from each library (μl x nM) was pooled from 16 randomized libraries to make the sequencing samples (total 2 pools of 16 libraries each) and the concentration of the pooled libraries was checked by Qubit. The library pools were denatured and diluted according to Illumina NextSeq protocols. The final concentration for sequencing was 2.5 pM + 2% Phix and it was run for 75 cycles SR (Single Read).