|
| Status |
Public on Jun 09, 2023 |
| Title |
Wound_CRISPR_cells |
| Sample type |
SRA |
| |
|
| Source name |
wounds that had been treated with hydrogels seeded with Ndrg2-knockout dendritic cells
|
| Organism |
Mus musculus |
| Characteristics |
passages: freshly isolated cells from wound tissue
tissue: skin wound
treatment: hydrogels seeded with Ndrg2-knockout dendritic cells
|
| Treatment protocol |
Dendritic cells were either treated with vitamin d3, CRISPR-knockout of Ndrg2 or left untreated, then submitted for scRNA-seq analysis. Wounds were treated with hydrogels seeded with Ndrg2-KO dendritic cells, control dendritic cells or blank hydrogels
|
| Growth protocol |
Freshly isolated murine bone marrow cells were cultured and differentiated into dendritic cells according to Lutz et al J Immunol Methods 223, 77-92 (1999).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Tissue was enzymatically digested, cells were collected and washed. These cellular suspensions were 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 single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Data processing |
Base calls were converted to reads using the Cell Ranger (10X Genomics; version 3.1) implementation of mkfastq
Then aligned against the mm9 (mouse) 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
Assembly: mm9 (mouse)
Supplementary files format and content: Cell Ranger output files
|
| |
|
| Submission date |
Jun 05, 2023 |
| Last update date |
Jun 21, 2023 |
| 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 |
GPL21103 |
| Series (1) |
| GSE234145 |
Cas9-Mediated Knockout of Ndrg2 Enhances the Regenerative Potential of Dendritic Cells for Wound Healing |
|
| Relations |
| BioSample |
SAMN35639669 |
| SRA |
SRX20599823 |
