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Sample GSM4042201 Query DataSets for GSM4042201
Status Public on Jun 28, 2022
Title sc_Donor_6_GEX
Sample type SRA
Source name Peripheral Blood
Organism Homo sapiens
Characteristics donor age: 46
donor gender: M
tissue: Peripheral Blood
cell type: CD8+ T-cells
pbmc type: Fresh
Growth protocol Fresh CD8+ cells were enriched from PBMC by using EasySep™ Human CD8 Positive Selection Kit II (Stem cells technologies) according to manufacturer’s instructions. Cells were resuspended in PBS containing 0.04% BSA. CD8+ cells were sorted as CD3+/CD19-/CD8+/CD4- cells into PBS containing 0.04% BSA. Purities of CD8+ T cells were 97 - 98%.
Extracted molecule total RNA
Extraction protocol For single-cell RNA seq library construction, cells were diluted to ~ 1X10^6/mL in 100 uL PBS containing 0.04% BSA and processed within hours after they were obtained. Briefly, freshly sorted CD8+ T cells were kept on ice before processing. Viable cells counted and about 8700 cells were used for Gel bead-in-emulsion (GEM) generation (targeted recovery ~5000 cells).
Single-cell RNA seq libraries were constructed using Single Cell 3’ library (v2 Chemistry) and Gel beads kits (10x genomics) according to the manufacturer’s protocol. After template switching reverse transcription in beads, cDNA was amplified in bulk. Subsequent fragmentation, end repair, A-tailing, adaptor ligation, and sample index PCR generated P5- and P7- capped, cell- and sample-indexed single-cell 3’ libraries. Libraries were quantified using both bioanalyzer (Agilent) and Kapa library quantification kit (Roche) before sequencing.
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 3000
Data processing Sequencing was performed on the Illumina HiSeq 3000/4000 flowcell (model #). Read 1, Read 2, and sample index were sequenced to 26, 98, and 8 basepairs, respectively.
The 10x Genomics cellranger 2.0.1 software was used to analyze raw base calls for this project. Cellranger MKFASTQ was used to demultiplex raw base calls by sample index. Cellranger COUNT was used to obtain UMI counts for each individual sample. Lastly, Cellranger AGGR was used to pool the individual samples together after depth normalization. It was noted that there were clusters of CD19+ and CD14+ in the default AGGR output. These two small clusters were thought to be comprised of B-lymphocyte and macrophage cells and were subsequently removed. Cells with above 2000 genes were thought to represent doublets in the drop-seq protocol and were removed.
The output from CellRanger AGGR was parsed by Seurat V3.0.0 as a cell barcode by gene matrix. Cells with mitochondrial DNA above 0.25% were removed.
Genome_build: Hg38 ENSEMBL 84
Supplementary_files_format_and_content: The processed data file, an .Rds file, was exported from the Seurat package. This file lists the cell barcodes (cell IDs) that remained after filtering out B cells, macrophages, and cells with more than 0.25% mitochondrial DNA. For each cell barcode, the associated donor number is listed as well as the UMI counts for each gene found in the cell.
Submission date Aug 22, 2019
Last update date Jun 28, 2022
Contact name Nan-Ping Weng
Organization name NIA
Department LMBI
Street address 251 Bayview Blvd
City Baltimore
ZIP/Postal code 21224
Country USA
Platform ID GPL21290
Series (1)
GSE136184 Single Cell Analysis of CD8+ T-cells with Aging
BioSample SAMN12619441
SRA SRX6750782

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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