GSM7963651
To perform RNA-seq on 3 month-old adult retinas, samd7stl888/+ heterozygotes were intercrossed to produce mixed genotype offspring and grown to adulthood. After adults were genotyped, pairs of retinas from three male WT and samd7stl888/stl888 zebrafish were dissected. The pair of retinas from an individual zebrafish were then combined to make one replicate, and RNA was extracted using the RNEasy Mini Kit (Qiagen) with on-column DNAse treatment using the Rnase-Free Dnase Set (Qiagen). RNA concentrations ranged from 20-37 ng/µl (600-1100 ng total), with RIN scores ranging from 9-9.5; there was minimal RNA degradation as shown by Bioanalyzer traces. Library preparation was performed with 10 ng of total RNA for 5-dpf whole eye and adult retina samples and with 300 pg of total RNA for 5-dpf thrb:tdTomato+ cells. Double-stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing (Takara-Clontech) per manufacturer's protocol using 12 amplification cycles for 5-dpf whole eye and adult retina samples, and 14 amplification cycles for 5-dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18, duty factor 20%, cycles per burst 50 for 120 seconds. cDNA was blunt-ended using a combination of T4 DNA Polymerase, Klenow Fragment DNA Polymerase, and T4 PolyNucleotide Kinase; an A base was added to the 3' ends using Klenow (3'-5' exo-), and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase (Qiagen-Enzymatics). Ligated fragments were then amplified for 12 cycles for 5-dpf whole eye samples, and 15 cycles for adult retina and 5-dpf thrb:tdTomato+ cell samples using primers incorporating unique dual-index tags, using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq-6000 using 150 bp paired-end reads.