GSM3354658: DMSO rep 2; Danio rerio; RNA-Seq
At 96 hpf, triplicate pools of 18-20 zebrafish eleutheroembryos exposed to either DMSO or 4,500 µg/L PCB-11 were transferred immediately to FastPrep homogenization tubes containing TRIzol Reagent (Life Technologies) and 0.1 mm RNase free Glass Beads (Next Advance, Inc.). The content was vortexed for 15 seconds, and homogenized in FastPrep-24 5G bead beater (MP Biomedicals) using the following parameters – speed: 6.0 m/sec, time: 30 seconds, and adapter: QuickPrep. The homogenate was spun, and the supernatant was transferred to a sterile tube. Total RNA was purified using the Direct-zol RNA MicroPrep kit with an in-column DNase-I treatment (Zymo Research Corp.), following manufacturer instructions. The quantity of RNA was assayed on Qubit using the RNA BR assay (Life Technologies Corp.), and quality was assessed on an Agilent 2100 Bioanalyzer using RNA 6000 Nano Assay (Agilent Technologies Inc.). The electropherogram results suggest that the total RNA was intact without any sign of RNA degradation, and the RNA Integrity Number (RIN) ranged from 6.9 to 9.2. One microgram of total RNA was used to isolate poly(A) mRNA, convert to cDNA and prepare libraries using the NEBNext Ultra-II Directional RNA Library Prep Kit for Illumina (New England Biolabs), following manufacturer instructions. The quantity of the library was assayed using the Qubit DNA HS assay (Life Technologies Corp), and quality was analyzed on a Bioanalyzer (Agilent Technologies Inc.). The libraries were of good quality as primer dimers or adapter dimers were not detected on the Bioanalyzer electropherogram. Libraries were diluted and normalized for equimolar amount, pooled, denatured and sequenced on an Illumina NextSeq 500 platform using NextSeq 500/550 High Output v2 kit (75 cycles) with 1×75 sequencing parameter. More than 32 million reads were generated per sample. RNA isolation, RNAseq library preparation and sequencing services were performed by the UMass Amherst Genomics Resource Laboratory, which acknowledges funding support from The Massachusetts Life Sciences Center (MLSC). Read alignment was performed with the BaseSpaceâ RNA-seq Alignment v.1.1.0 App, using the Danio rerio (UCSC danRer7) reference genome. Transcript assembly and differential expression was assessed with the BaseSpaceâ Cufflinks Assembly & DE v.2.1.0 App on the Illumina platform.