GSM7963641
To perform RNA-seq on 5-dpf larvae, samd7stl888/+ heterozygotes were intercrossed to produce mixed genotype offspring. Larvae were euthanized by exposure to tricaine, anterior halves of the body were collected in RNAlater (Invitrogen) and stored at 4°C, and corresponding posterior halves were collected in 50 mM NaOH for genotyping. Once genotypes were confirmed, eyes were dissected from the anterior body halves. The eyes from an individual larva constituted one replicate. Three WT and samd7stl888/stl888 replicates were collected and RNA-extracted using the RNeasy Micro Kit (Qiagen), then DNAse-treated and repurified using the Rneasy MinElute Cleanup Kit (Qiagen). RNA concentrations ranged from 1.5-2.2 ng/µl (~20 ng RNA total), with prominent 18/28S peaks and minimal RNA degradation in 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.