GSM8041943: AM633_Top_d100_Rep01 [AM6330028]; Danio rerio; RNA-Seq
Total RNA was isolated from zebrafish embryo samples from BioBide using the Qiagen RNeasy Mini kit following the manufacturer's protocol. RNA samples had RNA Quality Scores ≥7 as determined with a 5200 Fragment Analyzer System (Agilent, Santa Clara, CA). The Illumina 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 800 ng 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 double stranded cDNA fragments underwent end repair, poly A-tailing, and ligation to Illumina index-anchors.  Anchor-ligated library products were enriched with dual-indexed Illumina adapters (IDT for Illumina DNA/RNA Indexes sets) with 13 cycles of PCR. Individual sample libraries were analyzed for fragment quality and quantity using a Tapestation 4200 with D1000 screentape.  Individual libraries were pooled together based on equal nmol amount.  The pooled library was quantitated by Qubit fluorometer (Thermo Scientific, Waltham, Massachusetts, U.S.A.) and further diluted to 2 nM. The normalized and pooled library was further diluted to 2 pM and loaded onto an Illumina NovaSeq6000 following manufacturer's protocol and sequenced with single-read 100 bp configuration to generate raw RNAseq reads