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| Status |
Public on May 13, 2021 |
| Title |
Perifovea_donor_5 |
| Sample type |
SRA |
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| Source name |
retina
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| Organism |
Homo sapiens |
| Characteristics |
donor: donor_5
tissue: retina
region: parafovea
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| Extracted molecule |
total RNA |
| Extraction protocol |
A 1 mm foveal and 4 mm parafoveal punch of neural retina was acquired from human donors 5-8. After dissection away from the rpe and choroid, the retinal tissue was dissociated in 20 units/mL of papain with 0.005% DNase I (Worthington Biochemical Corporation, Lakewood NJ) on a shaker at 37 °C for 1 - 1.25 hours. 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.
Cryopreserved cells from single-cell donors 5-8 were rapidly thawed before barcoding with the 10X Genomics chromium system. Libraries were pooled and sequenced using the Illumina NovaSeq platform (San Diego, CA).
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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 |
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 GRCh38 with CellRanger (v3.0.1) using the CellRanger default human GTF and the following parameter: --expect-cells=8000.
Cells were filtered with the Seurat (v3.0.2) subset function. Cells with nUMIs less than 400 (to remove cells with poor read quality) or greater than 6500 (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: seurat_obj[["mito.genes"]] <- PercentageFeatureSet(seurat_obj, pattern = "^MT-")
For each of the eight libraries, reads were normalized with the Seurat (v3.0.2) NormalizeData function, and variable features were found with the following commands: my _object <- NormalizeData(my_object, normalization.method = "LogNormalize", scale.factor = 10000), my_object <- FindVariableFeatures(my_object, selection.method = “vst”, nfeatures = 2000)
Integration anchors from the first 25 dimensions of the canonical correlation analysis were used to integrate data with the following commands: retina.anchors <- FindIntegrationAnchors(object.list = my_object_list, dims=1:25), RETINA.combined <- IntegrateData(anchorset = retina.anchors, dims = 1:25)
Data scaling, principal component analysis, and clustering were performed with the following commands: RETINA.combined <- ScaleData(RETINA.combined, verbose = FALSE), RETINA.combined <- RunPCA(retina.combined, npcs = 30, verbose = FALSE), RETINA.combined <- RunUMAP(RETINA.combined, reduction = "pca", dims = 1:23), RETINA.combined <- FindNeighbors(RETINA.combined, reduction = "pca", dims = 1:23), RETINA.combined <- FindClusters(RETINA.combined, resolution = 0.35)
Genome_build: GRCh38
Supplementary_files_format_and_content: Processed expression data matrix files are provided in tab-delimited format for count(*_count.csv) and processed/normalized (*_normalized.csv) expression values. For processed/normalized files, log-normalized expression values (from GetAssayData(object = seurat_object)) were appended to relevant metadata (barcode, cluster label, and donor number from the manuscript). Each row represents a unique cell, and columns correspond to metadata and log normalized gene expression values. For count files, raw counts were obtained (from GetAssayData(object = seurat_object, slot = “counts”)) for each cell type in each cluster and appended to relevant metadata (barcode, cluster label, and donor number from the manuscript).
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| Submission date |
Mar 16, 2021 |
| Last update date |
May 13, 2021 |
| Contact name |
Andrew Voigt |
| Organization name |
University of Iowa
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| Street address |
375 Newton Road
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| City |
Iowa City |
| State/province |
Iowa |
| ZIP/Postal code |
52246 |
| Country |
USA |
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| Platform ID |
GPL24676 |
| Series (2) |
| GSE169047 |
Human photoreceptor cells from different macular subregions have distinct transcriptional profiles [single-cell RNA-seq] |
| GSE169048 |
Human photoreceptor cells from different macular subregions have distinct transcriptional profiles |
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| Relations |
| BioSample |
SAMN18321862 |
| SRA |
SRX10359212 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5175796_donor_5_perifovea_counts.csv.gz |
25.2 Mb |
(ftp)(http) |
CSV |
| GSM5175796_donor_5_perifovea_normalized.csv.gz |
30.4 Mb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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