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| Status |
Public on May 29, 2019 |
| Title |
kidney organoid, Day10_2 |
| Sample type |
SRA |
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| Source name |
Human pluripotent stem cells
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| Organism |
Homo sapiens |
| Characteristics |
cell type: Human pluripotent stem cells
tissue: Kidney organoid
developmental stage: Day10
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| Treatment protocol |
To generate 3D kidney organoids, day 10-12 differentiating cells were dissociated into single cells by Accutase. 2.5-5 x 104 cells (per well) were aggregated in basic differentiation media supplemented with 50 ng/mL FGF9 and 10 μM Y-27632 using round bottom ultra-low attachment 96-well plates (Corning). 24 hours later, Y-27632 was removed. To generate kidney organoids with a high glomerulus-to-tubule ratio, no patterning CHIR was needed. To generate kidney organoids with a high tubule-to-glomerulus ratio, 10 days patterning CHIR was applied from day 14 to day 24 of differentiation. 3D kidney organoids were transferred onto the upper chamber of transwell (Corning) for liquid-air interface culture on day 16-18 of differentiation (5-7 days after aggregation), with daily medium change at the bottom chamber. Taking H9 ESCs as an example, we seeded ~21,000 cells per cm2, and started differentiation when the cells reached 50-60% confluence. On day 10-12 of differentiation, we dissociated the differentiation culture into single cells, and re-aggregated into cell clusters with a gradient of cell numbers (5,000-75,000 cells per organoid). One million starting human ESCs could give rise to approximately 3,500 kidney organoids that were aggregated with 35,000 cells per organoid. hPSC-derived 3D kidney organoids were implanted beneath the renal capsule of left kidney of NSG mice for easier manipulation. After 2-4 weeks mice were sacrificed, and implants were isolated for further analysis.
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| Growth protocol |
Induction of kidney organoids from human pluripotent stem cells (hPSCs) hPSCs were dissociated into single cells with Accutase (STEMCELL Technologies), and plated onto Matrigel (BD Biosciences)-coated plates at a density of ~15,000 to ~24,000 cells/cm2. The plated cells were cultured in TeSR supplemented with 10 μM Y-27632 (Sigma) for 24 hours, followed by another 48-72 hours culture in TeSR to reach a confluence of 50-60%. To induce primitive streak, cells were cultured in basic differentiation medium (Advanced RPMI 1640, 1x L-GlutaMAX, 1 x NEAA) (Life Technologies) supplemented with 10 μM CHIR99021 (Sigma) for 4 days with daily media change. Next, to induce nephrogenic intermediate mesoderm, cells were cultured in basic differentiation medium, without any growth factor for 3 days with daily media change. Subsequently, to induce nephron progenitors, cells were cultured in basic differentiation media supplemented with 50 ng/mL FGF9 (Peprotech) and 3 μM CHIR99021 with daily media change for 2 days. From day 10 of differentiation onward, cells were cultured in basic differentiation media supplemented with 50 ng/mL FGF9 until day 20. On day 14 of differentiation, cells were pulsed with 1 μM CHIR99021 (patterning CHIR) for 24 hours. From day 20 of differentiation onward, basic differentiation medium was used for organoid culture.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from cells using Trizol (Invitrogen). RNA was purified using Quick-RNA kit (Zymo Research) with DNase treatment.
RNA samples of high integrity were used for RNA-seq library construction. rRNA depletion was conducted followed by library construction with NEBNext Ultra RNA Library Prep Kit. Constructed libraries were multiplexed and 150 bp paired-end sequenced with Illumina HiSeq 4000.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
Illumina Casava1.8 software used for basecalling.
Sequenced reads were mapped to Hg19 human genome using STAR v2.5.2a with parameters --outSAMtype BAM SortedByCoordinate --outSAMstrandField intronMotif --outSAMattributes NH HI AS nM NM MD --outSAMmapqUnique 50 --outBAMsortingThreadN 16 --outWigType wiggle --outWigStrand Stranded --outWigNorm RPM --outFilterType BySJout --outFilterMultimapNmax 1 --outFilterMismatchNmax 999 --outFilterMismatchNoverLmax 0.04 --outFilterIntronMotifs RemoveNoncanonical --alignIntronMin 20 --alignIntronMax 1000000 --alignMatesGapMax 1000000 --alignSJoverhangMin 8 --alignSJDBoverhangMin 1 --alignEndsType EndToEnd --quantMode TranscriptomeSAM GeneCounts
Genome_build: hg19
Supplementary_files_format_and_content: Cuffnorm output files with quartile normalized FPKM expression value of each sample replicate.
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| Submission date |
May 28, 2019 |
| Last update date |
May 29, 2019 |
| Contact name |
M.P. Philipott |
| Organization name |
Blizard Institute Barts and The London School of Medicine and Dentistry
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| Department |
Center for Molecular Medicine Cologne
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| Street address |
4 Newark St
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| City |
London |
| ZIP/Postal code |
E1 2AT |
| Country |
United Kingdom |
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| Platform ID |
GPL20301 |
| Series (2) |
| GSE131841 |
Generating Patterned Kidney Organoids for Studying Development and Diseases [bulk RNA-Seq] |
| GSE132026 |
Generating Patterned Kidney Organoids for Studying Development and Diseases |
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| Relations |
| BioSample |
SAMN11872840 |
| SRA |
SRX5908545 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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