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| Status |
Public on Nov 10, 2021 |
| Title |
Low-input, deterministic profiling of single-cell transcriptomes reveals individual intestinal organoid subtypes comprised of single, dominant cell types [C1-HEK] |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
High-throughput single-cell RNA-sequencing (scRNA-seq) has transformed our ability to resolve cellular properties across systems. A key scRNA-seq catalyzer was the introduction of microdroplet-based systems, which vastly improved sample handling and cell throughput. While powerful, the current microfluidic systems are limited to high cell density (>1000 cells) samples. This prevents the efficient processing of individual, small tissues or rare cells, leading to respectively confounded mosaic cell population read-outs or failed capture of diagnostically interesting cells. In this study, we developed a deterministic, mRNA-capture bead and cell co-encapsulation droplet system, DisCo, that overcomes these limitations by enabling precise particle position and droplet sorting control through combined machine-vision and multilayer microfluidics. We demonstrate that DisCo is capable of processing samples containing few cells (< 100 cells) at high efficiencies( >70%). To underscore the unique capabilities of DisCo, we mapped the developmental process of 31 individual intestinal organoids at the single cell level. This uncovered extensive cellular heterogeneity among organoids, revealing two so far uncharacterized organoid subtypes, “gobloids” and spheroids, predominantly consisting of respectively Muc2+ goblet and Ly6a+ stem cells. Further Disco data analysis thereby revealed strongly increased Yap1 target gene expression in these spheroids, suggesting mechano sensing as the underlying mechanism for their spontaneous formation. Together, our novel “no-cell-left-behind” platform enables the deterministic processing of input cells, allowing high-resolution snapshots of cellular heterogeneity among rare cells or individual, small tissues or organoids.Together, our novel “no-cell-left-behind” platform enables the deterministic processing of input cells, allowing high-resolution snapshots of cellular heterogeneity among rare cells or individual, small tissues or organoids.
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| Overall design |
To benchmark the cell recovery efficiency of the Fluidigm C1 platform, HEK 293T (ATCC Cat. No. SD-3515) cells were diluted to reach approximately 10, 20, 40 cells per µL.12 µL of cell solution was mixed with 8 µL of Suspension Reagent from Fluidigm. The obtained suspensions were generated separately from the same stock and then quantified in triplicate using microscopy by examining a volume of 2 x 2.5 µL of the suspension between two coverslips. Counts of all triplicates were averaged to estimate the cell input for 5 µL of Cell Mix to the C1 IFC. The experiment on the C1 machine was performed according to the kit’s manual: “SMART-Seq v4 Ultra Low Input RNA Kit for the Fluidigm C1 System, IFCs User Manual” (Clontech Laboratories, Inc.) using 10 – 17 µm 96-trap trap C1 IFC OpenApp chips. The prrotocol was run on SMART-Seq v4 (1861x/1862x/1863x) programs on the C1 machine. To verify successful loading and cell trapping, a Cell xCellence (Olympus) microscope was used. Final cDNA was quantified using the PicroGreen dsDNA assay and then tagmented using a Nextera XT kit.
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| Contributor(s) |
Bues J, Biočanin M, Pezoldt J, Dainese R, Chrisnandy A, Rezakhani S, Salen W, Gupta R, Amstad E, Claassen M, Lutolf M, Deplancke B |
| Citation missing |
Has this study been published? Please login to update or notify GEO. |
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| Submission date |
Sep 08, 2021 |
| Last update date |
Nov 10, 2021 |
| Contact name |
Marjan Biočanin |
| E-mail(s) |
marjan.biocanin@epfl.ch
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| Organization name |
EPFL
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| Department |
SV-IBI
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| Lab |
Laboratory for systems biology and genetics/UPDE
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| Street address |
Station 19, SV 3820
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| City |
Lausanne |
| State/province |
Vaud |
| ZIP/Postal code |
1015 |
| Country |
Switzerland |
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| Platforms (1) |
| GPL18573 |
Illumina NextSeq 500 (Homo sapiens) |
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| Samples (140)
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| This SubSeries is part of SuperSeries: |
| GSE148093 |
Low-input, deterministic profiling of single-cell transcriptomes reveals individual intestinal organoid subtypes comprised of single, dominant cell types |
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| Relations |
| BioProject |
PRJNA761691 |
| SRA |
SRP336185 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE183686_C1_HEK_Read_Count_Matrix.txt.gz |
2.4 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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