Low-input, deterministic profiling of single-cell transcriptomes reveals individual intestinal organoid subtypes comprised of single, dominant cell types [HEK Kinetic]

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. Overall design: A single-cell suspension of HEK 293T (ATCC Cat. No. SD-3515) was loaded on the system as described above, and the remaining volume stored on ice. To evaluate the temporal batch effect, cells were sampled from the same prepared suspension. After 20 minutes, droplets were evacuated from the system and sequencing libraries prepared. A new cell loading tip was inserted into the sample outlet port and the experimental run was resumed. The previous steps were repeated after 40 and 60 minutes. After 120 minutes, the system was loaded with cells stored on ice and cells captured for approximately 20 minutes. Subsequently, droplets were evacuated from the system, cDNA was generated, and sequencing libraries prepared as described above. The former steps were repeated 180 minutes from the first DisCo experiment.