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| Status |
Public on Jun 30, 2024 |
| Title |
Second_kidney_xeno_scRNA_seq_untransplanted |
| Sample type |
SRA |
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| Source name |
kidney
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| Organism |
Sus scrofa |
| Characteristics |
tissue: kidney
cell type: Dissociated kidney cells
treatment: subject 2, untransplanted
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Fresh kidney tissues were minced rapidly in a cell culture dish followed by washing with DPBS buffer. Minced tissue was resuspended in 5mL of 37°C pre-warmed 0.25% Trypsin for 20 min with mechanical dissociation using serological pipette every 5 min. Following digestion, dissociation mix was filtered through a 100µm strainer. The remaining large tissues on the strainer were further ground and washed to collect additional cells. Cell debris and dead cells were removed using Miltenyi Biotech Dead Cell Removal Kit before loading for 10X Genomics single-cell RNA-seq (v3.1 Chemistry). Longitudinal single-cell RNA-seq of the recipient’s PBMCs were prepared at the same time after collecting all samples across timepoints. Fresh PBMCs of each timepoint were first collected through gradient centrifugation, followed by cryopreservation of the prepared PBMCs according to the 10X Genomics protocol (protocol number: CG00039). Upon collecting all PBMC samples, the frozen cells were thawed at the same time, then loaded for 10X Genomics single-cell RNA-seq (v3.1 Chemistry).
prepared signle cells were sequenced using 10X Genomics single-cell RNA-seq (v3.1 Chemistry) following manufacturer's protocol.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Raw single-cell sequencing data were processed with the CellRanger pipeline of 10X Genomics to generate single-cell raw gene expression matrices. Kidney single-cell sequencing data were separately mapped to both human genome (GRCh38) and porcine genome (Sscrofa11) to distinguish the species origin of cells, while recipient’s PBMC single cell data were mapped to human genome (GRCh38) only. Single-cell raw expression matrices were then processed and analyzed using Seurat v4.3.049. Cells with less than 500 expressed genes, 1000 UMI, or more than 8% of transcripts from mitochondrial genes were removed from the kidney single cells, resulting in a total of 23,868 cells. A library of homologous genes between human and pig, obtained from the HGD Database50, was used as a reference to subset the filtered gene expression data for downstream analyses. Within the scope of gene expression of human-porcine one-to-one homologous genes, single cells with greater proportion of transcripts mapped to the porcine genome than the human genome are identified to be porcine cells, and vice versa to be human cells.
Each filtered dataset obtained from the kidneys and PBMC samples were normalized using SCTransform v2 from Seurat followed by integration using most commonly deemed variable features within each dataset. Dimensionality reduction analyses were performed on integrated kidney and PBMC datasets separately using Principal Component Analysis (PCA: npcs = 30) and Uniform Manifold Approximation and Projection (UMAP: dims = 1:30). Cell clusters were generated using the original Louvain algorithm with a resolution of 0.7 for integrated kidney dataset and 0.5 for the integrated PBMC dataset. Individual clusters were annotated based on the expression of cell type-specific markers.
Assembly: sscrofa11, hg38
Supplementary files format and content: h5
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| Submission date |
Feb 25, 2024 |
| Last update date |
Jun 30, 2024 |
| Contact name |
Weimin Zhang |
| E-mail(s) |
zhangweimindailab@gmail.com, weimin.zhang@nyulangone.org
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| Phone |
9175941528
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| Organization name |
NYU Langone Health
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| Department |
Institute for Systems Genetics
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| Lab |
Jef Boeke
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| Street address |
435 E 30th street
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| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10016 |
| Country |
USA |
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| Platform ID |
GPL26351 |
| Series (1) |
| GSE257542 |
Cellular dynamics of pig-to-human kdiney xenotransplantation [single cell RNA-seq] |
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| Relations |
| BioSample |
SAMN40120828 |
| SRA |
SRX23732156 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8107936_second_kidney_untransplanted_rt_Ssacrofa11_filtered_feature_bc_matrix.h5 |
19.8 Mb |
(ftp)(http) |
H5 |
| GSM8107936_second_kidney_untransplanted_rt_hg38_filtered_feature_bc_matrix.h5 |
10.9 Mb |
(ftp)(http) |
H5 |
SRA Run Selector |
| Raw data are available in SRA |
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