|
| Status |
Public on Feb 24, 2021 |
| Title |
Human 1 - No Strain (Control) |
| Sample type |
SRA |
| |
|
| Source name |
Dermal Fibroblasts
|
| Organism |
Homo sapiens |
| Characteristics |
individual: Human 1
passages: second to third passage
tissue: skin
cell type: Dermal Fibroblasts
treatment: No Strain (Control)
|
| Treatment protocol |
48 hours of 10% strain, or strain + 10uM FAKI
|
| Growth protocol |
Human skin samples were obtained under the approved IRB (#54225) and collected from three surgical procedures, fibroblasts were cultured and grown. Fibroblasts were then cultured in a 3D collagen scaffold system for 48 hours to either mechanically strain the cells or also treat with focal adhesion kinase inhibitor (FAKI)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
This cellular suspension was then submitted for droplet-based microfluidic single cell RNA sequencing (scRNA-seq) at the Stanford Functional Genomics Facility (SFGF) using the 10x Chromium Single Cell platform (Single Cell 3’ v3, 10x Genomics, USA).
cDNA was fragmented followed by end repair and A-tailing at 65°C for 30min. cDNA were double-sided size selected using SpriSelect beats. Sequencing adaptors were ligated to the cDNA at 20°C for 15min. cDNA was amplified using a sample-specific index oligo as primer, followed by another round of double-sided size selection using SpriSelect beads.
Final libraries were analyzed on an Agilent Bioanalyzer High Sensitivity DNA chip for qualitative control purposes. cDNA libraries were sequenced on a HiSeq 4000 Illumina platform aiming for 50,000 reads per cell.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Description |
KC-C
|
| Data processing |
Base calls were converted to reads using the Cell Ranger (10X Genomics; version 3.1) implementation of mkfastq
Then aligned against the GRCh38 v3.0.0 (human) genome using Cell Ranger’s count function with SC3Pv3 chemistry and 5,000 expected cells per sample
Cell barcodes representative of quality cells were delineated from barcodes of apoptotic cells or background RNA based on a threshold of having at least 200 unique transcripts profiled, less than 10,000 total transcripts, and less than 10% of their transcriptome of mitochondrial origin.
Unique molecular identifiers (UMIs) from each cell barcode were retained for all downstream analysis.
Raw UMI counts were normalized with a scale factor of 10,000 UMIs per cell and subsequently natural log transformed with a pseudocount of 1 using the R package Seurat (version 3.1.1).
Highly variable genes were identified, and cells were scaled by regression to the fraction of mitochondrial transcripts. Aggregated data was then evaluated using uniform manifold approximation and projection (UMAP) analysis
Genome_build: GRCh38 v3.0.0 (human)
Supplementary_files_format_and_content: barcodes.tsv.gz features.tsv.gz matrix.mtx.gz
|
| |
|
| Submission date |
Feb 23, 2021 |
| Last update date |
Feb 24, 2021 |
| Contact name |
Kellen Chen |
| E-mail(s) |
chen.kellen@gmail.com
|
| Organization name |
University of Arizona
|
| Department |
Surgery
|
| Street address |
1501 N Campbell Ave
|
| City |
Tucson |
| State/province |
AZ |
| ZIP/Postal code |
85724 |
| Country |
USA |
| |
|
| Platform ID |
GPL20301 |
| Series (1) |
| GSE167339 |
Disrupting Mechanotransduction Promotes Regenerative Phenotypes in Human Cells |
|
| Relations |
| BioSample |
SAMN18038075 |
| SRA |
SRX10156959 |
