|
| Status |
Public on Nov 22, 2023 |
| Title |
MSC and MEF-loaded Nanovials pooled scRNAseq |
| Sample type |
SRA |
| |
|
| Source name |
adipose/mesenchyme
|
| Organisms |
Homo sapiens; Mus musculus |
| Characteristics |
tissue: adipose/mesenchyme
cell line: ATCC SCRC-4000
cell type: Mesenchymal Stromal Cell/ Mouse Embryonic Fibroblast
|
| Treatment protocol |
MSCs were cultured and loaded onto gelatin coated nanovials for 2 hours before straining excess cells. Cells were bound and allowed to secrete for a 12 hour incubation. Nanovials are then collected and incubated with anti human VEGF-A antibody for 30 minutes. Nanovials containing single cells were isolated by FACS using calcein stain. Cell-loaded nanovials were then diluted and added directly to the single cell RNA-seq workflow.
|
| Growth protocol |
Immortalized human adipose-derived MSCs (ATCC SCRC-4000) were cultured in MSC Basal Medium supplemented with Low Serum MSC Growth Kit for adipose MSCs.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Nanovials were loaded directly into the cell mix at a concentration of 500 nanovials per ul for use in the 10x Chromium workflow.
Libraries were constructed as described in the Chromium Single Cell 3' Reagent Kits User Guide (v3.1 Chemistry Dual Index) with Feature Barcoding technology for Cell Surface Protein and Cell Multiplexing)
scRNAseq libraries were constructed as typically described in the guide above.
SEC-seq libraries proceeded via normal workflow, with inclusion of construction of a library to detect the oligo-barcoded antibody using the Feature Barcode library workflow in the Single Cell User guide above
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
MSC_MEF_MixedSpecies_Nanovials_scGEX
|
| Data processing |
Data was demultiplexed and output as FASTQ in Basespace using library indexes
FASTQs were mapped to their respective genomes using the Cellranger utility to construct a gene by cell matrix for each sample
Oligo barcodes (SEC-seq or nanovial hashing tags) were also mapped to cells using Cellranger multiconfig setup.
Gene by cell matrixes were processed and filtered in R using Seurat and basic utilities
For the mixed species experiment, the data was mapped to a joined human-mouse contig genome using Cellranger. Cells were assigned to each species based on the ratio of human to mouse reads
Assembly: GRCh38 (human), GRCm38 (mouse)
Supplementary files format and content: Data was filtered and normalized in Seurat. Nanovial barcodes and VEGFA barcodes are provided in metadata as raw unnormalized reads, while the cell-gene matrix is log transformed and normalized to cell depth.
Supplementary files format and content: For the purpose of filtering based on Nanovial barcodes, cell were removed from processed data which did not satisfy either barcode to barcode ratio cutoffs ("Mixed") or which had insufficient barcode impying separation from the nanovial
|
| |
|
| Submission date |
Jan 23, 2023 |
| Last update date |
Nov 22, 2023 |
| Contact name |
Kathrin Plath |
| Organization name |
University of California, Los Angeles
|
| Street address |
615 Charles E. Young Drive South
|
| City |
Los Angeles |
| State/province |
CA |
| ZIP/Postal code |
90095 |
| Country |
USA |
| |
|
| Platform ID |
GPL25526 |
| Series (1) |
| GSE223550 |
Secretion encoded single-cell sequencing (SEC-seq) uncovers gene expression signatures associated with high VEGF-A secretion in mesenchymal stromal cells |
|
| Relations |
| BioSample |
SAMN32881960 |
| SRA |
SRX19145063 |
