GSM8794304: wildtype whole eyes, 72 hours post fertilization, sample 3; Danio rerio; RNA-Seq
Whole eyes were dissected fromzebrafish at 72 hours post fertilization according to prior methods (Krall et al., 2018) We performed 'bulk' RNA-Seq experiments in dissected eyes from in-crossed crim1-/- larvae and controls at 72 hpf according to prior methods (Krall et al., 2018). RNA quality and quantity was reviewed with a bioanalyzer (2100 Bioanalyzer, Agilent) and libraries were prepared with an Ovation® RNA-Seq system V2 kit (NuGEN). With this method, total RNA was reverse transcribed to synthesize first-strand cDNA using a combination of random hexamers and a poly-T chimeric primer (Krall et al., 2018). The RNA template was then partially degraded by heating and the second strand cDNA was synthesized using DNA polymerase. The double-stranded DNA was then amplified using single primer isothermal amplification (SPIA). SPIA is a linear cDNA amplification process in which RNase H degrades RNA in DNA/RNA heteroduplex at the 5′-end of the double- stranded DNA, after which the SPIA primer binds to the cDNA and the polymerase starts replication at the 3′-end of the primer by displacement of the existing forward strand (Krall et al., 2018). Random hexamers were then used to amplify the second-strand cDNA linearly. Finally, libraries from the SPIA-amplified cDNA were made using the Ultralow DR library kit (NuGEN). The RNA-Seq libraries were analyzed for quality and primer dimers by bioanalyzer and quantified by quantitative polymerase chain reaction (qPCR) prior to sequencing (KAPA library quantification kit). High-throughput sequencing was done using single-end 50 lane(s) on a HiSeq 4000 instrument (Illumina).