RNA-Seq of Danio rerio: pooled 96 hpf larvae
Larval zebrafish, 96 hours post fertilization, were collected from 4 to 5 biological replicates, larval samples pooled, and stored in RNAlater until RNA extractions. Larvae were homogenized using plastic pestles and total RNA extracted using an RNeasy Mini kit (Qiagen). A Qubit Fluorometer was used to measure RNA concentrations with RNA quality and integrity assessed on an Agilent 2100 Bioanalyzer. Samples used for downstream library preparation had high quality RNA Integrity Numbers (RIN > 8). RNA was diluted to 500 ng and used in the NEBNext Poly(A) mRNA Magnetic Isolation Module. Generated mRNA was used in the NEBNext Ultra II Directional RNA library Prep Kit (New England Biolabs) to generate cDNA, while utilizing primers from NEBNext Multiplex Oligos for Illumina. An Agilent 2100 High Sensitivity Bioanalyzer was used to assess cDNA for proper fragment size. Single-end, 1 x 75 bp sequencing was performed on an Illumina NextSeq500 high-throughput sequencer.