GSM5510108: can4-/- (homo) rep5 (16281X15); Danio rerio; RNA-Seq
We first incrossed heterozygous can4 mutants to produce a mixture of can4+/- (heterozygous), can4-/- (homozygous), and can4+/+ (wild type; WT) embryos. We then lysed 3 dpf larvae individually by rigorously pipetting in 55 µl DNA/RNA Shield (Zymo Research) at RT followed by overnight incubation at 4°C. We extracted genomic DNA from 5 µl lysate of each larvae using Zymo 96 genomic DNA purification kit and genotyped each sample by PCR and Sanger sequencing. The remaining lysates were stored at -80°C while awaiting genotyping results. Once genotyped, larvae with the same genetic background were pooled for mRNA extraction using Direct-zol RNA Miniprep Kit with TRI Reagent from Zymo Research. We prepared 5 biological replicates for each genetic background – can4-/- and WT – with each sample containing mRNA extracted from 9 larvae. Libraries were prepared using the Illumina TruSeq Stranded Total RNA Library Prep Ribo-Zero Gold Kit.