GSM5961901: zfish, embryo, 72hpf, 10d; Danio rerio; RNA-Seq
Total RNA was isolated from pooled samples of 8-10 embryos (four pools for each exposure) using the TRIzol reagent protocol with modifications (Invitrogen, Carlsbad, CA). Briefly, TRIzol was added to frozen embryos, before allowing embryos to thaw. Samples were homogenized in a Bead Mill 4 (Fisher Scientific, PA) using sterile 2 mm glass beads. RNA was resuspended in 50 µL RNase-free water. Quantity and quality of samples were determined using a NanoDrop 2000 (A260/280 and A260/230 > 1.8). Between 500 – 750 ng of total RNA per sample were sent to the Scripps Research Institute Next Generation Sequencing Core (TSRI, San Diego, CA) for shallow RNA-sequencing. cDNA library preparation and shallow RNA-Seq was performed at TSRI. Sample quality was assessed using a 2100 Bioanalyzer 2100 (Agilent Technologies) (RIN>8.5). Library preparation for RNA-Seq was performed using the HTP RNA-Seq Library Prep Kit (iGenomX Inc, San Francisco, CA). Briefly, barcoded oligo dT primers were added to 50 ng RNA per sample, and reverse transcribed. Sample cDNA products were combined, cleaned, and run through PCR with 0.5 µM barcoded PCR primers (p5 and p7 sequences, Illumina, San Diego, CA).  Purified PCR products were sequenced using a NextSeq2000 sequencer (Paired-end mode; read1: 26 bases, read2: 94bp) at TSRI. Greater than 1.5 million paired-end reads were generated for each sample.