Deterministic scRNA-seq of individual intestinal organoids reveals new subtypes and coexisting distinct stem cell pools

While Single-cell RNA-sequencing (scRNA-seq) has transformed our ability to resolve cellular properties across systems, current microfluidics-based scRNA-seq technologies are limited to samples with large amounts of cells (> 1,000 cells). This prevents efficient processing of individual, small tissue samples, leading to confounded mosaic cell population read-outs. To overcome these limitations, we developed a etermintic, mRNA-capture bead and cell -encapsulation dropleting system, DisCo, that enables precise particle and cell positioning and droplet sorting control through combined machine-vision and multilayer microfluidics. To underscore the unique capabilities of our approach in processing low-input samples (< 100 cells) at high efficiency (> 70%), we analyzed intestinal organoid development by “DisCo-ing” 31 individual organoids at varying developmental stages. This revealed extensive organoid heterogeneity, uncovering a so far uncharacterized “gobloid” subtype consisting predominantly of precursor and mature (2) goblet cells. We also identified spheroids that are comprised of a secondary regenerative fetal-like stem cell population, marked by prolonged YAP1 nuclear translocation and target gene expression, and that persist as symmetrical cysts even under differentiation conditions. These findings illustrate the power of our “no-cell-left-behind” platform in providing high-resolution snapshots of cellular heterogeneity among individual, small tissues.