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GEO help: Mouse over screen elements for information. |
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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 [Intestinal crypts] |
| Organism |
Mus musculus |
| 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 |
Crypts were isolated from single small intestines of 7-week old male C57BL/6J mice following the protocol from Bas and Augenlicht et al. Briefly, the small intestine of one mouse was isolated and then washed both on the inside and outside with ice cold PBS. The small intestine was cut open longitudinally and washed again with PBS. The intestine was then non-enzymatically digested for 3 minutes in PBS/EDTA/DTT. Next, the tissue was cut into small pieces and transferred into a 50 mL Falcon tube containing 20 mL of ice-cold PBS. The PBS solution containing the tissue pieces was gently triturated 10 times using a 10 mL pipette. After tissue fragments had sedimented, the supernatant was removed, and the process was repeated three more times until the supernatant was clear. Next, the supernatant was removed, PBS/EDTA was added and the sample incubated for 30 minutes at 4oC on a rocking plate. After incubation, tissue fragments were left for sedimentation (up to 5 minutes), then the supernatant was removed. Subsequently, tissue fragments were triturated with ice cold PBS by pipetting up and down. After large tissue fragments sedimented (up to 5 minutes), the supernatant containing crypts was collected as Fraction 1 (F1). Fraction collection was repeated five times (F2-F5), followed by trituration with ice cold PBS, while each fraction was stored separately. Each fraction was inspected for cell debris and villus contamination.For single-cell bulk sample preparation, crypts from F2 or F3 were spun down at 600 x g for 10 minutes (brake 5). Following centrifugation, the supernatant was removed and cells were enzymatically dissociated for 1 minute at 37oC. Cells were then washed two times in PBS/BSA and strained two times using a Flowmi 40 µm strainer to minimize the doublet rate. Cell suspensions were diluted in cell loading buffer and applied to the loading tip connected to the DisCo chip. For single-cell isolation from single crypts, crypts from F3 were transferred to FBS coated 6 well plates. Subsequently, single crypts were isolated by hand-picking after which they were transferred to a Nunc microwell culture plate containing single crypt dissociation mix. Single crypts were dissociated by combining trituration using non-filter pipette tips every 5 minutes and incubation at 37oC for a total of 15 minutes. Following dissociation, cell suspensions were diluted in cell loading buffer and applied to the loading tip connected to the DisCo chip.
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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 12, 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) |
| GPL19057 |
Illumina NextSeq 500 (Mus musculus) |
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| Samples (27)
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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 |
PRJNA761688 |
| SRA |
SRP336192 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE183691_RAW.tar |
4.3 Mb |
(http)(custom) |
TAR (of TXT) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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