Bead-free deterministic DNA barcoding using vacuum-driven loading of aqueous oligonucleotides to microwell arrays.

Achieving high throughput in experiments requiring sample indexing depends primarily on the precise and reproducible deposition of reagents prior to analysis. Contemporary droplet and microwell systems utilize random deposition of oligonucleotide-coated beads into reaction chambers, requiring costly bead synthesis and offering limited control over the final distribution of barcoded beads. As an alternative, we present deterministic, aqueous barcoding of 512 arrayed microwells using a multi-layer, vacuum-driven microfluidic network. To uniquely barcode each of the 512 microwells, we deposit DNA oligonucleotide solutions designed using a Combinatorial Dual Indexing (CDI) (i5, i7) scheme via deterministic loading. Deterministic fluid loading is achieved by sequentially coupling two bifurcated, orthogonal microchannel networks with a planar microwell array. The microchannel networks actuate fluid flow through a combination of an applied vacuum force and a dead-end channel design. After loading the oligonucleotide solutions, we observed uniform barcode patterning across the arrays of microwells (∼20% CV), reasonable barcode loading times (30-40 min per step), and reduced reagent use (∼8-16 µL at 25 µM oligos vs. 10-50 µL at 100 µM for bead systems). We detected cross-contamination in ∼4% of the microwells. Following DNA barcode delivery, we utilized the platform to generate high-quality ATAC-seq libraries for breast cancer cell line (MCF7) nuclei by integrating tagmentation and on-chip PCR. Overall, we describe a deterministic and bead-free DNA barcoding strategy for efficient barcoding of microwell arrays that are important in single-cell analyses.