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| Status |
Public on May 17, 2019 |
| Title |
Fovea donor 1 |
| Sample type |
SRA |
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| Source name |
Retina
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| Organism |
Homo sapiens |
| Characteristics |
location: Fovea
donor: Donor 1
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| Extracted molecule |
total RNA |
| Extraction protocol |
A 2-mm foveal centered and a 4-mm peripheral punch from the inferotemporal region were acquired from three clinically normal human donors. Tissue was dissociated using the Papain Dissociation System (Worthington Biochemical Corporation, Lakewood NJ). Dissociated cells were resuspended in DMSO-based Recovery Cell Culture Freezing Media (Life Technologies Corporation, Grand Island NY). Suspensions were placed in a Cryo-Safe cooler (CryoSafe, Summerville SC) to cool at 1°C/minute in a -80°C freezer for 3-8 hours before storage in liquid nitrogen.
Single-cell RNA libraries were prepared for sequencing using standard 10X genomics protocols. Briefly, cryopreserved samples were thawed, and single cells were captured and barcoded using the Chromium System with the v3 single cell-reagent kit (10x Genomics, Pleasanton CA). Sequencing was performed on pooled libraries using the Illumina HiSeq 4000 platform (San Diego, CA), generating 150 base pair paired-end reads.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
FASTQ files were generated from the raw BCL files using Illumina’s bcl2fastq conversion program.
Sequenced reads were mapped to the CellRanger human genome build hg19 (v3.0.0) with CellRanger (v3.0.1) using the CellRanger default human GTF and the following parameter: --expect-cells=8000.
The six samples were collectively aggregated with the cellranger aggr function with the following parameter: --normalized=mapped.
Cells were filtered with Seurat (v2.3.4) FilterCells function. Cells with nUMIs less than 200 (to remove cells with poor read quality) or greater than 2500 (to remove cells likely to be doublets) were removed. Cells with greater than 60% of reads originating from mitochondrial genes were also removed. Mitochondrial genes were identified with the following command: mito.genes <- grep(pattern = "^MT-", x = rownames(x = seurat_object@data), value = TRUE).
Aggregated reads were normalized with Seurat (v2.3.4) with the following command: NormalizeData(object = seurat_object, normalization.method = "LogNormalize", scale.factor = 10000). Variable genes were identified from downstream normalization with: FindVariableGenes(object = seurat_object, mean.function = ExpMean, dispersion.function = LogVMR, x.low.cutoff = 0.0125, x.high.cutoff = 3, y.cutoff = 0.5). Cell-to-cell variation regression was performed to minimize any batch effect introduced in the sample preparation process with: ScaleData(object = seurat_object, vars.to.regress = c("nUMI", "percent.mito")).
Clustering was performed with Seurat (v2.3.4) FindClusters. In order to generate the shared nearest neighbor (SNN) graph, the principal component analysis reduction technique was utilized for the first 10 principal components. A granularity resolution value of 0.6 was used to discriminate clusters.
Genome_build: hg19
Supplementary_files_format_and_content: Processed expression data matrix files are provided in tab-delimited format. Log-normalized expression values (from the seurat_object@data slot) were appended to relevant metadata (barcode and cluster label from the manuscript). Each row represents a unique cell, and columns correspond to metadata and log normalized gene expression values.
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| Submission date |
May 02, 2019 |
| Last update date |
Dec 20, 2019 |
| Contact name |
Todd Scheetz |
| E-mail(s) |
todd-scheetz@uiowa.edu
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| Organization name |
UNIVERSITY OF IOWA
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| Street address |
3181B MERF
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| City |
Iowa City |
| State/province |
IA |
| ZIP/Postal code |
52242 |
| Country |
USA |
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| Platform ID |
GPL20301 |
| Series (1) |
| GSE130636 |
Single-cell RNA-Seq Investigation of Foveal and Peripheral Expression in the Human Retina |
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| Relations |
| Reanalyzed by |
GSE142449 |
| BioSample |
SAMN11566806 |
| SRA |
SRX5783051 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3745992_fovea_donor_1_expression.tsv.gz |
1.6 Mb |
(ftp)(http) |
TSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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