Zebrafish Embryonic Development Hypoxia Versus Normoxia RNAseq: Sample       60hpf-Normoxia
We collected whole RNA from zebrafish at five different embryonic developmental stages with experimental triplicates for each stage (time-point) and experimental condition.  For hypoxia, we placed developing embryos in pre-conditioned media in a sealed plexiglass chamber set to a reduced oxygen concentration for 12 hour periods. Immediately following hypoxia (or normoxia) embryos were sacrificed and RNA collected.  We used 1% pO2 for ages up to 48 hpf (hours post-fertilization); and 4% pO2 for older ages.  At the time-point for collection embryos were dissected and only head tissue was used for subsequent RNA preparation.  RNA was prepared with a miRNeasy kit (Qiagen). Quality of total RNAs was checked using a NanoDrop 2000 Spectrometer and an Agilent 2100 Bioanalyzer.  The sequencing libraries were created using the Illumina TruSeq Stranded Total RNA Sample Preparation Kit with Ribo-Zero. These libraries were sequenced on an Illumina HiSeq2000 sequencer for 50 cycles (ie. 50 nucleotides, single end, stranded) using Illumina’s version 3 chemistry. 6 samples were multiplexed together in each lane of the sequencer, providing an average yield of 25.1 million reads per sample. The base calling was performed using Illumina’s CASAVA version 1.8.2 software.