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

Single-cell RNA-sequencing (scRNA-seq) has transformed our ability to resolve cellular properties across systems. However, current scRNA-seq platforms are one-size-fits-all approaches that are tailored toward large cell inputs (> 1,000 cells), rendering them inefficient and costly when processing small, individual tissue samples. This important drawback tends to be resolved by loading bulk samples, but this yields confounded mosaic cell population read-outs. To overcome these technological limitations, we developed a deterministic, mRNA-capture bead and cell co-encapsulation dropleting system, DisCo. We demonstrate that DisCo enables precise particle and cell positioning and droplet sorting control through combined machine-vision and multilayer microfluidics. In comparison to other microfluidics systems, the active flow control driving DisCo, enables continuous operation and processing of low-input samples (< 100 cells) at high capture efficiency (> 70%). To underscore the unique capabilities of our approach, we analyzed intestinal organoid development by "DisCo-ing" 31 individual organoids at varying developmental stages. This revealed extensive organoid heterogeneity, identifying distinct subtypes including a regenerative fetal-like Ly6a+ stem cell population which persists as symmetrical cysts even under differentiation conditions. Furthermore, we uncovered a so far uncharacterized "gobloid" subtype consisting predominantly of precursor and mature (Muc2+) goblet cells. These findings demonstrate the unique power of DisCo in providing high-resolution snapshots of cellular heterogeneity among small, individual tissues.