Combinatorial perturbation sequencing on single cells using microwell-based droplet random pairing

Combinatorial drug therapy reduces drug resistance and disease relapse, but informed drug combinations are lacking due to the high scale of possible combinations and the relatively simple phenotyping strategies. Here we report combinatorial perturbation sequencing (CP-seq) on single cells using microwell-base droplet random pairing. CP-seq uses oligonucleotides to barcode drugs, encapsulates drugs and cells in separate droplets, and pairs cell droplets with two drug droplets randomly on a microwell array chip to complete combinatorial drug treatment and barcode-tagging on cells. The subsequent single-cell RNA sequencing simultaneously detects the single-cell transcriptomes and drug barcodes to demultiplex the corresponding drug treatment. The microfluidic droplet operations had robust performance, with overall success rate among the microwells being up to 83%. We then progressively validated the CP-seq by performing single drug treatment and then combinatorial drug treatment. Leveraging the advantage of droplet microfluidics in massive multiplexing, the CP-seq can test thousands of drug combinations in a single experiment and represents a great technology for combinatorial perturbation screening with high throughput and comprehensive profiling.