|
| Status |
Public on Jul 18, 2024 |
| Title |
Right kidney, Sham operated, 24h, rep1, snRNAseq |
| Sample type |
SRA |
| |
|
| Source name |
cross sectional sample
|
| Organism |
Mus musculus |
| Characteristics |
tissue: Right kidney
tissue: cross sectional sample
strain: C57BL/6
treatment: Sham operation
time post_surgery: 24h
treatment: Sham operation
gender: Male
mouse id: 71
batch: 3
age: 8-12 weeks
library kit: 10x Genomics Chromium Next GEM v3.1, Library & Gel Bead Kit
bp size: 100
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Nuclei were isolated from a section of the kidney as per published protocol (Leiz et al., 2021). Briefly, kidney section was minced and homogenized in EZ lysis buffer. The homogenate was filtered through a 40 µm cell strainer and centrifuged to pellet the nuclei. The nuclei pellet was resuspended and layered onto a sucrose gradient and centrifuged to separate and remove any other cellular components and debris. The purified nuclei were resuspended in an appropriate volume of resuspension buffer. Single nuclei were then sorted based on DAPI fluorescence using a flow cytometer. The sorted nuclei were counted using a CellDrop FL Automated Cell Counter (DeNovix) to determine the concentration. The single nuclei suspension was loaded onto a Chromium Controller (10x Genomics) for single nuclei encapsulation, barcoding and cDNA synthesis.
Library preparation using the Chromium Single Cell 3' Reagent Kit v3 (10x Genomics) following the manufacturer's instructions. The prepared libraries were sequenced on an MGI FCL sequencer.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
DNBSEQ-G400 |
| |
|
| Description |
Sham71
Sham71
|
| Data processing |
Raw sequencing data were processed using the Alevin-fry pipeline with the mouse reference genome (mm10) splice reference and index for pseudo-alignment and gene quantification was carried out using Alevin-fry's quant mode. The resulting gene expression matrix was imported into Seurat for single-cell RNA sequencing data analysis. Seurat was used for quality control, filtering out low-quality cells and potential doublets. The filtered data were then SCTransform normalized and to regress out percentage of mitochondria and ribosomal transcript per cell. Principal component analysis (PCA) was performed on the variable genes, followed by unsupervised clustering using the Louvain algorithm. Harmony was used to remove the influence of batch effects11 and Uniform Manifold Approximation and Projection (UMAP) was employed for dimensionality reduction and visualization of the clusters. Differential gene expression analysis was performed on individual clusters using Seurat's FindMarkers function. The results were used to identify marker genes and characterize the cell types present in the kidney tissue.
mm10
csv files; meta data and information for each nucleus labelled by sample name followed by cell barcode.
csv files; raw gene counts from Alevin-fry pipeline output for each nucleus labelled by sample name followed by cell barcode.
|
| |
|
| Submission date |
Aug 18, 2023 |
| Last update date |
Jul 18, 2024 |
| Contact name |
Jonathan Gleadle |
| E-mail(s) |
jonathan.gleadle@flinders.edu.au
|
| Organization name |
Flinders University
|
| Department |
College of Medicine and Public Health
|
| Lab |
Renal lab
|
| Street address |
3D226 Renal Lab, Flinders Medical Centre, Flinders Drive
|
| City |
Bedford Park |
| State/province |
SA |
| ZIP/Postal code |
5042 |
| Country |
Australia |
| |
|
| Platform ID |
GPL28457 |
| Series (1) |
| GSE241213 |
Gene expression profile at bulk (24h, 48h & 72h) and single nucleus (24h) level of mouse right kidney post sham operation and unilateral nephrectomy. |
|
| Relations |
| BioSample |
SAMN37051590 |
| SRA |
SRX21417433 |
