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| Status |
Public on Feb 18, 2025 |
| Title |
Urine cells, membranous nephropathy patient, ADT |
| Sample type |
SRA |
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| Source name |
urine
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| Organism |
Homo sapiens |
| Characteristics |
tissue: urine
patient: membranous nephropathy patient
library type: adt
adt: TotalSeq-B0062 anti-human CD10, TotalSeq-B0364 anti-human CD13, TotalSeq-B0180 anti-human CD24, TotalSeq-B0073 anti-human CD44, TotalSeq-B0952 Streptavidin (binding anti-human CD133 biotin (miltenyi 130-113-669), TotalSeq-B0123 anti-human CD326
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| Extracted molecule |
protein |
| Extraction protocol |
Samples were collected either as first morning void urine or via urinary catheter (using the pooled urine output of 4 hours) and stored on ice. Incubation with Actinomycin D for 30 min on ice was applied to prevent subsequent alteration of transcriptomes followed by blocking Fc receptors using human FcR blocking reagent (Miltenyi, Bergisch Gladbach, Germany). Cells were then incubated with fluorescent dyes and fluorochrome-conjugated monoclonal antibodies for fluorescence-activated cell sorting (FACS). The following antibodies and dyes were used for cell labeling: Calcein AM (BD Biosciences, San Jose, CA, USA, 564061), Hoechst 33342 (BD Biosciences, San Jose, CA, USA, 561908), AnnexinV-PE (Biolegend, San Diego, CA, USA, 640908), CD45-APC/Vio770 and CD66b-PE/Vio770 (both Miltenyi, 130-110-635 and 130-119-768). Cells were washed, filtered through a 70 µm cell strainer and labeled with To-Pro3 iodide (T3605, Life technologies, Eugene, Oregon) for dead cell discrimination immediately prior to sorting. Samples were sorted using a Sony MA900 (Sony, Tokyo, Japan) with a 100µm nozzle into phosphate buffered saline (PBS) / 1 % BSA gating To-Pro3-AnnexinV- Hoechst33342+CalceinAM+ singlets for obtaining single viable urinary cells. Urinary granulocytes, which make up a large fraction of the urinary cellular signal (median 58% in FACS gating) but for which a renal tissue origin is not verifiable, and which may interfere with downstream analysis, were excluded. Relative single cell counts in sorted suspensions were counted using a MACSQuant Analyzer (Miltenyi). Cells were centrifuged, resuspended, and re-counted on a hemocytometer. Cell suspensions were subjected to single-cell sequencing following the 10x Genomics protocol for Chromium Next GEM Single Cell v3.1 chemistry targeting between 1000 - 10000 cells depending on sample cell count.
According to manufacturer recommendation of Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1
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| Library strategy |
OTHER |
| Library source |
other |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
fastqc to check quality of sequencing reads
cellranger (v3.1) mkfastq to demultiplex fastq files
cellranger (v3.1) count to align sequencing files to a reference transcriptome
Assembly: Assembly: GRCh38
Supplementary files format and content: provide library.csv and feature_ref.csv for cellranger (v3.1) count of ADT samples
Supplementary files format and content: Tab-separated UMI counts in matrix.mtx. List of genes in features.tsv List of cell barcodes in barcodes.tsv
Library strategy: CITE-seq
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| Submission date |
Nov 19, 2024 |
| Last update date |
Feb 18, 2025 |
| Contact name |
Jan Klocke |
| E-mail(s) |
jan.klocke@charite.de
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| Organization name |
Charité Universitätsmedizin Berlin
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| Department |
Nephrology and Intesive Care
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| Street address |
Charitéplatz 1
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| City |
Berlin |
| ZIP/Postal code |
10117 |
| Country |
Germany |
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| Platform ID |
GPL20301 |
| Series (1) |
| GSE282344 |
Flow-cytometric quantification of urine kidney epithelial cells specifically reflects tubular damage in acute kidney diseases |
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| Relations |
| BioSample |
SAMN44826298 |
| SRA |
SRX26771107 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8641668_NR_JK_051_barcodes.tsv.gz |
13.0 Kb |
(ftp)(http) |
TSV |
| GSM8641668_NR_JK_051_genes.tsv.gz |
93 b |
(ftp)(http) |
TSV |
| GSM8641668_NR_JK_051_matrix.mtx.gz |
52.0 Kb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
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