title: RNA-seq of Danio rerio: 32 hpf embryo
design: 15 embryos from each treatment (CV6, CV24, or GF) were randomly selected at 32 hpf, pooled, and flash frozen in liquid nitrogen (experiments repeated four times). RNA was extracted from each pooled embryo sample using RNeasy QIAGEN Mini Kit (74104, QIAGEN Inc.) according to the manufacturers instructions and concentration and quality was analyzed using a NanoDrop UV-Vis Spectrophotometer (Thermo Fisher Scientific, Inc., Waltham, MA). 200 ng RNA was used to generate KAPA mRNA HyperPrep (Roche, Basel, Switzerland) whole-transcriptome libraries. RNA was captured with magnetic oligo-dT beads and fragmented at 94C for 2 minutes. Libraries were barcoded using Illumina Dual Index Adapters (Illumina, Inc., San Diego, CA) and amplified by PCR for 14 cycles. Amplified libraries were then pooled and the Illumina NovaSeq 6000 system (Illumina, Inc., San Diego, CA) was used for 50 base pair paired-end sequencing.