|
| Status |
Public on Aug 25, 2021 |
| Title |
Peripheral blood mononuclear cell - P2 |
| Sample type |
SRA |
| |
|
| Source name |
Peripheral blood
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Peripheral blood
|
| Growth protocol |
Organoids were maintained in growth factor-reduced matrigel and grow in progenitor tumor organoid media (PaTOM)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Biopsies were minced with sterile surgical scalpels to 0.5-1 mm fragments in approximately 1 ml of media. After centrifugation for 5 minutes at 125 g, cells were resuspended in DMEM with 0.5 mg/ml Liberase TH (Sigma-Aldrich) and 1% penicillin-streptomycin (Corning) and incubated in an orbital shaker for 15 min at 37°C. Liberase was quenched with equal volume of 1% BSA in DMEM followed by centrifugation for 5 minutes at 125 g. Cells were resuspended in 2 ml Accutase (Sigma-Aldrich) and incubated in an orbital shaker for 15 minutes at 37°C. Organoids were dissociated by incubating with TrypLE Express (Thermo Fisher) for 5-15 minutes at 37°C followed by manual disruption. Dissociated cells were passed through a 40 μm strainer and centrifuged for 5 minutes at 125 g. Isolated cells were resuspended in 0.04% BSA in phosphate buffered saline (Corning) for subsequent viability analysis and counting.
Single-cell transcriptomic amplification and library preparation was performed using the 5’ or 3’ Library Construction Kit (10x Genomics) following the manufacturer’s recommendations. Amplification and library prep of the PM sample was performed using the SureCell WTA 3’ Library Prep Kit for the ddSEQ System following the manufacturer’s recommendations (Illumina, Cat# 20014279).
single cell RNA sequencing
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
PBMC-P2
|
| Data processing |
Raw base call (BCL) files were demultiplexed and converted to FASTQ files, which were subsequently used to generate feature-barcode matrices using Cell Ranger RNA v3.1 (10x Genomics). PM sample was run through the Single cell RNA-seq app on BaseSpace. Briefly, reads were aligned against a reference genome using Spliced Transcripts Alignment to a Reference (STAR), followed by barcode tagging and BAM indexing. Count file was generated using gene UMI counter and with cells passing quality filter based on cells above background and passing knee filter.
Initial single-cell analysis was performed using Seurat v3.1.0 (2). Cells containing less than 200 genes were removed, and log-normalized expression of 2,000 variable genes were used to reduce the data into two-dimensional space.
Cells from all 9 biopsies were combined using FindIntegrationAnchors and IntegrateData functions. Cell identities were manually assigned using highly expressed genes. To retain the highest number of cells for subsequent analyses, no additional filters were initially applied.
Genome_build: hg19
Supplementary_files_format_and_content: filtered_feature_bc_matrix, UMI count file in CSV format
|
| |
|
| Submission date |
Aug 18, 2020 |
| Last update date |
Aug 25, 2021 |
| Contact name |
Anirban Maitra |
| E-mail(s) |
[email protected]
|
| Organization name |
The University of Texas MD Anderson Cancer Center
|
| Department |
Translational Molecular Pathology
|
| Lab |
Maitra laboratory
|
| Street address |
6565 MD Anderson Blvd
|
| City |
Houston |
| State/province |
TX |
| ZIP/Postal code |
77030 |
| Country |
USA |
| |
|
| Platform ID |
GPL18573 |
| Series (1) |
| GSE156405 |
Elucidation of tumor-stromal heterogeneity and the ligand-receptor interactome by single cell transcriptomics in real-world pancreatic cancer biopsies |
|
| Relations |
| BioSample |
SAMN15848232 |
| SRA |
SRX8961827 |
