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Status |
Public on Dec 16, 2021 |
Title |
KR02 |
Sample type |
SRA |
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Source name |
iPSC-derived kidney organoids without SARS-CoV-2 infection
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Organism |
Homo sapiens |
Characteristics |
tissue: Kidney organoid condition: CTR
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Treatment protocol |
To study direct effects of the virus on kidney cells independent of systemic effects we chose human induced pluripotent stem cell (iPSC)-derived kidney organoids to model SARS-CoV-2 infection in vitro, using the Bavarian strain of the virus (MT270101.1). iPSC-derived kidney organoids were infected with SARS-CoV-2 at an MOI of 1.0 at d7+18. Virus incubation lastet 4h until changing the medium back to E6 without virus and incubating the organoids for five more days.
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Growth protocol |
To generate human induced pluripotent stem cell (iPSC) derived kidney organoids, iPSCs were seeded using a density of 18,000 cells/cm2 on Geltrex (Thermo Fisher, Breda, the Netherlands) coated 6-well plates (Greiner Bio-one, Alphen aan de Rijn, the Netherlands). The differentiation protocol was modified from Takasato et al. (Takasato et al. 2016). On day 0 (d 0), differentiation was initiated using CHIR-99021 (6 µM, R&D systems, Abingdon, United Kingdom) in Essential 6 medium (E6, Thermo Fisher). CHIR treatment was maintained for 3 or 5 days and medium was replaced sequentially for E6 supplemented with fibroblast growth factor 9 (FGF9), 200 ng/ml, R&D systems) and heparin (1 µg/ml, Sigma-Aldrich, Zwijndrecht, Netherlands) up to d 7. Cell layers were trypsinized and suspensions were counted on d 7. One part 3 days CHIR-differentiated cells was mixed with two parts 5 days CHIR-differentiated cells. To generate cell aggregates, cells were aliquoted using 300,000 cells per 1.5 ml tube and centrifuged. Cell aggregates were plated on Costar Transwell filters (type 3450, Corning, Sigma-Aldrich). One hour CHIR pulse (5 µM) in E6 was used to stimulate self-organizing nephrogenesis and the medium was replaced for E6 supplemented with FGF9 and heparin for additional 5 days. As of d 7+5, organoids were cultured using E6 media supplemented with human epidermal growth factor (hEGF, 10 ng/ml, Sigma-Aldrich), bone morphogenetic protein-7 (BMP7, 50 ng/ml, R&D systems), stromal derived factor 1 beta (SDF1β, 10 ng/ml, R&D systems), vasopressin (10 nM, Sigma-Aldrich) and aldosterone (10 nM, Sigma-Aldrich) until viral infection at d 7+18.
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Extracted molecule |
total RNA |
Extraction protocol |
All protocols to generate scRNA-seq data on the 10x Chromium platform, including sample prep, library prep, instrument and sequencing settings, are available here: https://support.10xgenomics.com/single-cell-gene-expression
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina NovaSeq 6000 |
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Data processing |
The Cell Ranger Software Suite (version 3.1.0) was used for processing sequencing information and single cell barcodes. Seurat (version 3.2.2) was used to integrate the scRNA-seq data and predict cell labels. Genome_build: GRCh38 Supplementary_files_format_and_content: barcodes: barcodes present in input data Supplementary_files_format_and_content: matrix: a count matrix with Market Exchange Format (MEX). The first line contains the type code and the second line includes format and cell-ranger versions. Number of peaks, barcodes, and non-zero entries can be found in the third line. The rest of the lines represent nonzero entries with the first two numbers corresponding to peak and barcode indices and the last one corresponding to the number of cut sites. Supplementary_files_format_and_content: genes: a bed file contains genes corresponding to row indices. For each gene, its gene ID and name are stored in the first and second column of the gene.tsv file, respectively
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Submission date |
Feb 26, 2021 |
Last update date |
Dec 16, 2021 |
Contact name |
Ivan Gesteira Costa |
E-mail(s) |
ivan.costa@rwth-aachen.de
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Phone |
+49 241 80 89337
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Organization name |
UKA RWTH Aachen
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Department |
Joint Research Center for Computational Biomedicine
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Lab |
Institute for Computational Genomics
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Street address |
Pauwelsstr. 19
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City |
Aachen |
State/province |
Northrhine-Westphalia |
ZIP/Postal code |
52074 |
Country |
Germany |
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Platform ID |
GPL24676 |
Series (1) |
GSE167747 |
SARS-CoV-2 infects the human kidney and drives fibrosis in kidney organoids |
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Relations |
BioSample |
SAMN18075507 |
Supplementary file |
Size |
Download |
File type/resource |
GSM5112198_KR02_barcodes.tsv.gz |
56.3 Kb |
(ftp)(http) |
TSV |
GSM5112198_KR02_features.tsv.gz |
325.6 Kb |
(ftp)(http) |
TSV |
GSM5112198_KR02_matrix.mtx.gz |
74.7 Mb |
(ftp)(http) |
MTX |
Raw data not provided for this record |
Processed data provided as supplementary file |
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