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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Feb 20, 2024 |
| Title |
Renal LEC, saline, scRNAseq |
| Sample type |
SRA |
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| Source name |
kidney
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| Organism |
Mus musculus |
| Characteristics |
tissue: kidney
cell type: Lymphatic endothelial cells
strain: C57BL6/J
age: 10-12 weeks of age
treatment: saline
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| Extracted molecule |
total RNA |
| Extraction protocol |
Kidneys were minced into small pieces and placed into 5mL digestion buffer (15mg/mL Collagenase D (Roche Applied Science, 11088858001), 10mg/mL Collagenase Type 4 (Gibco, 17-105-019), 2 mg/mL Dispase II (Roche Applied Biosciences, 4942078001), BSA Fraction V (Fisher Scientific, BP1605-100), and HBSS (ThermoFisher Scientific, 14025092). The samples were then incubated in a 37°C water bath with manual agitation every 10 minutes. Digestion was halted by adding 10mL of ice cold HBSS. Digested samples were filtered through both 70µm and 40 µm filters. Endothelial cells were enriched magnetically using a mouse CD31 biotinylated antibody (BD Pharmingen, 553371) and a cleavable Biotin Binder Kit (Invitrogen, 11533D). After the linker was cleaved, the isolated cells were subsequently enriched using an anti-mouse podoplanin (Pdpn) biotinylated antibody (BAF3244, R&D Systems) as above. CD31+Pdpn+ cells were placed into ice cold RPMI 1640 + 10% FBS prior to FACS confirmation of CD45-CD31+Pdpn+ cells. Cell fractions were resuspended in in RPMI1640 +10% FBS, counted, and diluted to a concentration of ~1,000 viable cells/μL.
Library preparation was performed in accordance with the manufacturer's instructions. Renal LEC samples were mixed with master mix, paritioning oil, and gel then loaded onto the Chromium X Controller using the Chromium Next GEM Single Cell 3’ Reagent Kit V3.1. Sample cDNA quality was checked using the Agilent High Sensitivity D5000 ScreenTape assay on the Agilent 4200 TapeStation System (Agilent Technologies, G2991BA). cDNA samples were then quantified and normalized prior to the library sequencing. Libraries were sequenced on a NovaSeq S4 Flow cell according to 10x Genomics recommendations.
scRNA-seq. Libraries were sequenced on a NovaSeq S4 Flow cell according to 10x Genomics recommendations.
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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 |
Sample demultiplexing, barcode processing, and gene count were conducted with CellRanger V6.0.1. Briefly, single-cell RNA sequencing reads were processed using 10x Genomics cloud analysis. Briefly, fastq files were uploaded to the 10x Genomics Analysis cloud and aligned to the reference mouse transcriptome (mm10) using Cell Ranger Count v6.0.1. a digital gene expression matrix, including raw unique molecular identifier (UMI) counts were assigned to each cell in the respective sample. (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
Assembly: mm10
Supplementary files format and content: Tab-separated values files and matrix files
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| Submission date |
Jun 29, 2023 |
| Last update date |
Feb 20, 2024 |
| Contact name |
Joseph M. Rutkowski |
| Organization name |
Texas A&M University School of Medicine
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| Department |
Medical Physiology
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| Street address |
8447 Riverside Parkway
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| City |
Bryan |
| State/province |
Texas |
| ZIP/Postal code |
77807 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE236199 |
Single-cell RNA sequencing identifies response of renal lymphatic endothelial cells to acute kidney injury |
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| Relations |
| BioSample |
SAMN36083375 |
| SRA |
SRX20830535 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7519638_saline_barcodes.tsv.gz |
3.0 Mb |
(ftp)(http) |
TSV |
| GSM7519638_saline_features.tsv.gz |
254.1 Kb |
(ftp)(http) |
TSV |
| GSM7519638_saline_matrix.mtx.gz |
17.2 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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