RNA-Seq Blog Transcriptome Research & Industry News
Single-cell RNA-seq and single-cell assay for transposase-accessible chromatin (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 multiome assays, custom droplet microfluidics may provide solutions complementary to commercial setups.
Researchers at VIB-KU Leuven 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 noncommercial scATAC-seq protocol in droplets. After validating HyDrop-ATAC, they applied it to flash-frozen mouse cortex and generated 7996 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 9508 single-cell transcriptomes closely matching reference single-cell gene expression data. Finally, they leveraged HyDrop-RNA’s high capture rate to analyze a small population of fluorescence-activated cell sorted neurons from the Drosophila brain, confirming the protocol’s applicability to low input samples and small cells. HyDrop is currently capable of generating single-cell data in high throughput and at a reduced cost compared to commercial methods, and they envision that HyDrop can be further developed to be compatible with novel (multi) omics protocols.
Technical overview of HyDrop barcoded bead production and quality control.
(a) Split-pool process for barcoding of dissolvable hydrogel beads for single-cell RNA-sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin (scATAC-seq). Unbarcoded hydrogel beads are sequentially distributed over 96 wells, sub-barcoded using linear amplification polymerase chain reaction (PCR), repooled, and re-distributed two more times to generate 96 × 96 × 96 (884,736) possible barcode combinations. Different 3-prime terminal capture sequences are possible depending on the oligonucleotide sequence appended in the last step. (b) Semiquantitative assessment of bead primer incorporation by fluorescence in-situ hybridisation (FISH) using a fluorescein amidite (FAM) probe after every sub-barcoding step shows that bead fluorescence uniformity is retained throughout the barcoding process. (c) FISH with FAM 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 nonfluorescent beads
