HyDrop enables droplet based single-cell ATAC-seq and single-cell RNA-seq using dissolvable hydrogel beads

Single-cell RNA-seq and single-cell ATAC-seq technologies are used extensively to create cell type atlases for a wide range of organisms, tissues, and disease processes. To increase the scale of these atlases, lower the cost, and pave the way for more specialized multi-ome assays, custom droplet microfluidics may provide solutions complementary to commercial setups.

Researchers at VIB-KU Leuven Center for Brain and Disease Research have developed HyDrop, a flexible and open-source droplet microfluidic platform encompassing three protocols. The first protocol involves creating dissolvable hydrogel beads with custom oligos that can be released in the droplets. In the second protocol, the researchers demonstrate the use of these beads for HyDrop-ATAC, a low-cost non-commercial scATAC-seq protocol in droplets. After validating HyDrop-ATAC, they applied it to flash-frozen mouse cortex and generated 7,996 high-quality single-cell chromatin accessibility profiles in a single run. In the third protocol, they adapt both the reaction chemistry and the capture sequence of the barcoded hydrogel bead to capture mRNA, and demonstrate a significant improvement in throughput and sensitivity compared to previous open-source droplet-based scRNA-seq assays (Drop-seq and inDrop). Similarly, the researchers applied HyDrop-RNA to flash-frozen mouse cortex and generated 9,508 single-cell transcriptomes closely matching reference single-cell gene expression data. Finally, they leveraged HyDrop-RNA’s high capture rate to analyse a small population of FAC-sorted neurons from the Drosophila brain, confirming the protocol’s applicability to low-input samples and small cells.

Split-pool process for barcoding of dissolvable hydrogel beads. Beads are sequentially distributed over 96 wells, sub-barcoded, re-pooled, and distributed three times to generate 96✕96✕96 (884736) possible barcode combinations. Different 3′-terminal capture sequences are possible depending on the oligonucleotide sequence appended in the last step. b. Semi-quantitative assessment of bead primer incorporation by FISH after every sub-barcoding step shows that bead fluorescence uniformity is retained throughout the barcoding process. c. FISH with probes complementary to only one of 96 sub-barcode possibilities shows that approximately 1/96 beads exhibit fluorescence for a selected sub-barcode probe. Fluorescence signal is overlaid with a brightfield image at 50% transparency to indicate positions of non-fluorescent beads. d. Microfluidic chip setup on the Onyx platform. Cells and beads are loaded into pipette tips and plugged into a HyDrop Chip. Flow of oil and aqueous phases is achieved by Onyx displacement syringe pumps. e. HyDrop chip design has three inlets: one each for carrier oil, barcoded hydrogel beads and cell/reaction mix. Passive filters at each inlet prevent dust and debris from entering the droplet generating junction. f. Diagram and snapshot of cell/bead droplet encapsulation. Schematic overview of HyDrop-ATAC (g) and HyDrop-RNA (h) assay for single-cell library generation. Nuclear membrane is visualised in salmon, water droplet is visualised in blue.

HyDrop is currently capable of generating single-cell data in high throughput and at a reduced cost compared to commercial methods, and the developers envision that HyDrop can be further developed to be compatible with novel (multi-) omics protocols.