GSM7963659
To perform RNA-seq on 5-dpf thrb:tdTomato+ cells, the following crosses were performed: WT × WT;thrb:tdTomato+/- or samd7stl888/stl888 × samd7stl888/stl888;thrb:tdTomato+/-. A subset of larvae were genotyped from each resultant clutch. TdTomato+ larvae were then separated for subsequent dissociation. 40-50 eyes from 20-25 larvae were dissected from each clutch as an individual replicate. The eyes were then dissociated into single cells and subjected to FACS. 10,000-25,000 cells were collected and sorted directly to buffer RLT from the Rneasy minElute Cleanup Kit (Qiagen); RNA was then extracted using the Rneasy Micro Kit (Qiagen), and on-column DNAse treatment performed using the Rnase-Free Dnase Set (Qiagen). RNA concentrations ranged from 40-190 pg/µl (0.3-1.8 ng total), with RNA integrity (RIN) scores ranging from 7.4-8.4. 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.