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| Status |
Public on Jun 06, 2024 |
| Title |
Rpe65-KO TMB |
| Sample type |
SRA |
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| Source name |
retina
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| Organism |
Mus musculus |
| Characteristics |
tissue: retina
genotype: Rpe65-KO
time: 2 months
treatment: TMB pellet
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| Treatment protocol |
Dietary vehicle (normal pellets) or TMB treatment was started at P21-P24.
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| Growth protocol |
All mice were housed under a standard 12-hour light (≤150 lux)/12-hour dark cycle, fed ad libitum, and maintained using standard procedures.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Eyes were enucleated for retinal tissue isolation. After removing the anterior chamber and lens, the retina was dissected from the posterior eye cup. Then, the retinal cells were dissociated using the Papain Dissociation System (#LK003153, Worthington Biochemical) following the manufacturer's instructions.
Sublibraries were generated and sequenced independently on Illumina NovaSeq6000 at an average sequencing depth of 50,000 reads/cell, for a projected total of 5 billion reads for the entirety of the 100,000 cell barcoded library. Reads from identical cells from each of the sublibraries were consolidated during analysis and processing of raw reads.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
RPE65_KO_TMB
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| Data processing |
The pre-processing steps such as generation and demultiplexing of FASTQ files from raw sequencing reads (bclfastq, v2.20), aligning to UCSC mm10 transcriptome, and generating raw count matrices were conducted using Cell Ranger (v6.0.1) with default parameters. Cumulus software v1.5.0 was used to combine expression matrices and to remove cell doublets by Scrublet v0.2.1. The pipelines were run in Terra Cloud Platform (https://app.terra.bio/). Seurat v3.2.2 was used to perform downstream analysis following the standard pipeline using cells with >=500 genes, resulting in 4,135 WT mouse cells, 3,979 vehicle treated RPE65 cells, and 5,901 TMB treated RPE65 cells. Principal Component (PC) analysis was performed on a submatrix of the top 1,000 most variable genes computed using the function FindVariableGenes in Seurat. Batch effect between experimental conditions was minimzed using the Harmony package v0.1.0 to remove non-cell-type-specific factors that may impact clustering. The number of top PCs was assessed by the elbow method. Cells were clustered using a shared nearest neighbor modularity optimization-based algorithm (FindClusters in Seurat).
Assembly: mm10
Supplementary files format and content: ExpressionMatrix.mtx.gz: Expression Matrix is sparse format.
Supplementary files format and content: MetaData.csv.gz: Metadata related to columns (cells) of the Expression Matrix.
Supplementary files format and content: Genes.txt.gz: Gene symbols related to rows of the Expression Matrix.
Supplementary files format and content: CellIDs.txt.gz: Cellular barcodes in the Expression Matrix.
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| Submission date |
Apr 12, 2024 |
| Last update date |
Jun 06, 2024 |
| Contact name |
Marcin Tabaka |
| E-mail(s) |
mtabaka@ichf.edu.pl
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| Organization name |
International Centre for Translational Eye Research
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| Lab |
Tabaka Lab
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| Street address |
Kasprzaka 44/52
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| City |
Warsaw |
| ZIP/Postal code |
01224 |
| Country |
Poland |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE263877 |
A combination treatment based on drug repurposing demonstrates mutation-agnostic efficacy in pre-clinical retinopathy models [scRNA-seq: Rpe65-KO] |
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| Relations |
| BioSample |
SAMN40945984 |
| SRA |
SRX24236635 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8200464_RPE65_KO_TMB_feature_bc_matrix.h5 |
46.3 Mb |
(ftp)(http) |
H5 |
SRA Run Selector |
| Raw data are available in SRA |
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