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Sample GSM4042211

Query DataSets for GSM4042211

Status

Public on Jun 28, 2022

Title

sc_Donor_16_GEX

Sample type

SRA

Source name

Peripheral Blood

Organism

Homo sapiens

Characteristics

donor age: 90
donor gender: F
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

E-mail(s)

wengn@mail.nih.gov

Organization name

NIA

Department

LMBI