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| Status |
Public on Jun 21, 2024 |
| Title |
thrb:tdTomato+ cells from 5-dpf larval eyes, samd7-/-, rep2 |
| Sample type |
SRA |
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| Source name |
eye
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| Organism |
Danio rerio |
| Characteristics |
cell type: thrb:tdTomato+ cells
tissue: eye
developmental stage: 5-dpf larvae
genotype: samd7-/-
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| Extracted molecule |
total RNA |
| Extraction protocol |
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.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Sequencing adapters were trimmed using trimgalore 0.6.1 (https://github.com/FelixKrueger/TrimGalore)61. RNA-seq reads were then aligned to danRer10 using STAR 2.7.2b with an index prepared for 150 bp reads62. Next, Htseq 0.9.1 was used to generate normalized read counts63. To calculate differential gene expression, DESeq2 1.34.0 was used in R 4.1.3, using a log2 fold-change threshold of 0 and an FDR (p-adj) of 0.164. Volcano plots were then labeled to identify genes with p-adj < 0.05. To identify genes as photoreceptor subtype-specific, manual curation was performed using publicly available RNA-seq data from adult zebrafish photoreceptor subtypes4,5. To identify rod-specific-gene dysregulated in the samd7-/- adult retina (Table S3), we manually cross-referenced the top 40 most enriched rod-specific genes and mafba from Ogawa et al5.
Assembly: danRer10
Supplementary files format and content: csv file of DESeq2-normalized counts and averages from three samd7-/- and WT replicates, derived from 5dpf thrb:tdTomato+ cells. Genes are ranked by p-adj.
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| Submission date |
Dec 08, 2023 |
| Last update date |
Jun 21, 2024 |
| Contact name |
Joseph Corbo |
| E-mail(s) |
jcorbo@wustl.edu
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| Phone |
(314) 362-7787
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| Organization name |
Washington University School of Medicine
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| Department |
Pathology and Immunology
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| Street address |
660 S. Euclid
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| City |
Saint Louis |
| State/province |
MO |
| ZIP/Postal code |
63110 |
| Country |
USA |
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| Platform ID |
GPL24995 |
| Series (2) |
| GSE249755 |
Samd7 preserves cell identity and enforces the ‘one neuron-one receptor’ rule in vertebrate photoreceptors [5dpf_td] |
| GSE249756 |
Samd7 preserves cell identity and enforces the ‘one neuron-one receptor’ rule in vertebrate photoreceptors |
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| Relations |
| BioSample |
SAMN38730852 |
| SRA |
SRX22832955 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
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