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| Status |
Public on May 12, 2022 |
| Title |
Kidney organoids 5-25mM oscillatory glucose 1dpi SARS-CoV-2 |
| Sample type |
SRA |
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| Source name |
Human Kidney Organoid
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| Organism |
Homo sapiens |
| Characteristics |
tissue: kidney organoid
treatment: 5-25mM oscillatory glucose every 12h
infection: SARS-CoV-2 infection
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| Treatment protocol |
hPSCs grown on vitronectin were washed twice with PBS (Life Technologies #1001-015) and dissociated into small clusters with EDTA (Sigma #E9884). Cells were then seeded onto vitronectin pre-coated culture dishes at a density of 50.000-150.000 cells per cm2 in Essential 8 Medium. After overnight culture, the differentiation was initiated (day -4) by treatment with 8µM CHIR99021 (Sigma #SML1046-5MG) in basic differentiation medium [Advanced RPMI 1640(Life technologies #12633-012) + 1 × 1-GlutaMAX (Life Technologies #35050-038) + Penicillin/Streptomycin (Life Technologies #15140122)] for 3 days. On day -1, CHIR99021 was removed by washing once with PBS and cells were exposed to 200 ng ml-1 FGF9 (PeproTech House#100-23B), 1 µ ml -1 Heparin (Sigma#H3149-10KU) and 10 ng ml-1 Activin A (Vitro S.A #338-AC-050) in basic differentiation media for 24h. On day 0, cells were mechanically dissociated into single cells and resuspended in basic differentiation medium supplemented with 3μM CHIR99021, 200 ng ml-1 FGF9 and 1µg ml-1 Heparin. Cells were then placed in 96-well, round bottom, ultra-low attachment plates at a density of 500.000 cells per well. Cells were spun down at x300g for 2 min form a pellet and then cultured at 37ºC, 5% CO2 for 2 days additional days without media change. On day 2, the pellets were transferred onto a Transwell 0.4µm pore polyester membrane (Sigma#3460) and treated with basic differentiation media supplemented with 3μM CHIR99021, 200 ng ml-1 FGF9 and 1µg ml-1 Heparin. On day 3, CHIR99021 was removed and the organoids were switched to basic differentiation media supplemented with 200 ng ml-1 FGF9 and 1µg ml-1 Heparin for another 4 days. From that point, organoids were cultured in basic differentiation medium and maintained without additional growth factors. For experiments employing glucose challenge, day 16 kidney organoids were diexposed to low glucose DMEM (ThermoFisher) supplemented with 1% Penicillin-Streptomycin for 6h (starvation period). After this time, normoglycemic conditions were assessed exposing organoids into DMEM supplemented with 5mM D-Glucose changing medium every day until day 23. Hyperglycaemic-like organoids were incubated with DMEM supplemented with 5mM D-Glucose on even days or with 25mM of D-Glucose on odd days until day 23 (5-25mM oscillatory hyperglycaemic condition).
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| Growth protocol |
hPSCs were maintained in Essential 8 medium (Life Technologies #A1517001) in p10 culture plates coated with vitronectin (Fisher Scientific S.L. A14700) in a 37ºC incubator with 5% CO2 and were passaged every 5-6 days.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
After the indicated treatments kidney organoids were collected and washed twice with PBS, and further dissociated into a single cell suspension by treating them with Accumax (07921, Stem Cell Technologies) for 15 min at 37ºC followed by Trypsin-EDTA 0.25% (wt/vol) trypsin (25300-054, Life Technologies) for additional 15 min at 37ºC. The reaction was deactivated by adding 10% FBS. The cell suspension was then passed through a 40μm cell strainer. After centrifugation at 1,000 RPM for 5 minutes cell numbers and viability were analyzed using Countess Automated Cell Counter (Invitrogen). This method generated single cell suspension with greater than 90% viability. Cell suspensions were frozen until further use. For library construction cells were thawed and centrifuged at 1,000 RPM for 5 minutes. Cell numbers and viability were analyzed using Countess Automated Cell Counter (Invitrogen) and cell suspensions were loaded onto a well of a 10X Chromium Single Cell instrument. Barcoding and cDNA synthesis were performed according to the manufacturer´s instructions. The cDNA libraries were constructed using the 10X Chromium Single cell 3' Library Kit v3 according to the manufacturer's original protocol and sequenced either with a NovaSeq 6000 (R1:28,R2:94,i7:8) or a NextSeq 500 (R1:28, R2:55, i7:8) instrument.
RNA libraries were prepared for sequencing using standard Illumina protocols
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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 |
Demultiplexing, alignment to the Homo sapiens (hg38) genome and quantification using the annotation from Ensembl v93 was performed using 10X Genomics Cell Ranger (v4.0.0)
Downstream analysis steps (quality control filtering, normalization, integration, clustering and differential expression) were performed using the R package Seurat (v3.2.1)
Genome_build: hg38
Supplementary_files_format_and_content: HDF5 Feature Barcode Matrix containing a Gene x Cells UMI count matrix
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| Submission date |
Jul 28, 2021 |
| Last update date |
May 12, 2022 |
| Contact name |
Asier Ullate-Agote |
| E-mail(s) |
[email protected]
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| Organization name |
CIMA
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| Department |
Hemato-Oncology
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| Lab |
Advanced Genomics
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| Street address |
Av. de Pío XII, 55
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| City |
Pamplona |
| State/province |
Navarra |
| ZIP/Postal code |
31008 |
| Country |
Spain |
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| Platform ID |
GPL24676 |
| Series (1) |
| GSE181002 |
Single-cell RNA-seq of human kidney organoids |
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| Relations |
| BioSample |
SAMN20456207 |
| SRA |
SRX11582736 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5481912_25mM_SARS_filtered_feature_bc_matrix.h5 |
51.4 Mb |
(ftp)(http) |
H5 |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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