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. We 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, we demonstrate the use of these beads for HyDrop-ATAC, a low-cost non-commercial scATAC-seq protocol in droplets. After validating HyDrop-ATAC, we 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, we 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, we applied HyDrop-RNA to flash-frozen mouse cortex and generated 9,508 single-cell transcriptomes closely matching reference single-cell gene expression data. Finally, we 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. HyDrop is currently capable of generating single-cell data in high throughput and at a reduced cost compared to commercial methods, and we envision that HyDrop can be further developed to be compatible with novel (multi-) omics protocols.
The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE175684 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE175684) and on SCope (https://scope.aertslab.org/#/HyDrop/*/welcome).Source Data files have been provided for figures 2a, 3a, 3b, 3c and 3i, and can be regenerated data analysis tutorials for HyDrop.Data analysis tutorials for HyDrop are available on GitHub (https://github.com/aertslab/hydrop_data_analysis).
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Animal experimentation: All animal experiments were conducted according to the KU Leuven ethical guidelines and approved by the KU Leuven Ethical Committee for Animal Experimentation (approved protocol numbers ECD P037/2016, P014/2017, and P062/2017). All use of cell lines was approved by the KU Leuven Ethical Committee for Research under project number S63316.
© 2022, De Rop et al.
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