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

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    Evaluation Summary:

    This paper introduces a flexible microfluidics-based single-cell genomics technology that expands and improves over existing custom droplet-based scRNA-seq protocols (inDrops and Drop-seq) in important ways: better data quality, simplified workflow, high cell recovery, and flexibility towards other single-cell applications, as exemplified by HyDrop-based single-cell ATAC-seq. Its flexibility should allow the research community to develop and implement new and custom workflows on this platform, including single-cell multi-omics technologies.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

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Abstract

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. 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 noncommercial scATAC-seq protocol in droplets. After validating HyDrop-ATAC, we 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, 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 9508 single-cell transcriptomes closely matching reference single-cell gene expression data. Finally, we 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 we envision that HyDrop can be further developed to be compatible with novel (multi) omics protocols.

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  1. Evaluation Summary:

    This paper introduces a flexible microfluidics-based single-cell genomics technology that expands and improves over existing custom droplet-based scRNA-seq protocols (inDrops and Drop-seq) in important ways: better data quality, simplified workflow, high cell recovery, and flexibility towards other single-cell applications, as exemplified by HyDrop-based single-cell ATAC-seq. Its flexibility should allow the research community to develop and implement new and custom workflows on this platform, including single-cell multi-omics technologies.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

  2. Reviewer #1 (Public Review):

    Droplet-based single-cell method development has stalled in the past years. The field has been overtaken by commercial solutions that optimized performance, but at much higher costs and without any possibility for customization. More recently, combinatorial indexing methods (e.g. SPLIT-seq) have gained popularity, requiring no specialized equipment and offering the possibility to massively increase output, but at the cost of reduced sensitivity.

    De Rop et al. introduce a flexible microfluidics-based single-cell genomics technology that expands and improves over previously existing custom droplet-based scRNA-seq protocols (inDrops and Drop-seq) in several directions: better data quality, simplified workflow, high-cell recovery, and flexibility towards other single-cell applications, as exemplified by HyDrop-based single-cell ATAC-seq.

    This is a much welcome development in the field and one that will hopefully stir the further development and optimization of custom droplet-based single-cell protocols (multi-ome, scChIP, and beyond).

  3. Reviewer #2 (Public Review):

    The authors present HyDrop as a flexible open-source and non-commercial microfluidic platform for scATAC and scRNA-seq using custom dissolvable hydrogel beads loaded with barcoded oligos. Optimisation, quality assurance and benchmarking experiments show that HyDrop performs well compared to inDrop and Drop-seq platforms. Moreover, the manuscript contains new datasets generated with this platform to show its sensitivity and use in studying cellular heterogeneity in a range of model organisms. Its flexibility should allow the research community to develop and implement new and custom workflows on this platform, including single-cell multi-omics technologies.

    Strengths:

    1. This manuscript describes the development of three protocols for an open-source droplet-microfluidics based single-cell genomics platform, HyDrop. The protocols are described in detail in the materials and methods and in the accompanying documentation on protocols.io.
      Small note to make here is that the source of some reagents is not explicitly mentioned. This is particularly important, for instance, for the source of the transposase used in the workflows. This should be remedied to enhance the ease of adoption and to avoid confusion.
    2. The set of experiments designed to test the performance of the described methods was very well designed and included explanations for the rationale behind the choices made, and were convincingly executed. The conclusions on his performance are well supported by the presented evidence, which is clearly described and explained. Importantly, inclusion of these 'technical' data allows the readers to assess the quality for themselves in the context that is most relevant to them.
    3. The applications of HyDrop for scATAC and scRNA-seq on human, mouse and drosophila samples support the notion of a robust platform that should be broadly applicable in other areas as well.
    4. The use of a single microfluidic chip design for both scATAC and scRNA-seq is elegant. At the same time the 3-inlet chip (cells, beads and oil, akin to the 10X design) may limit some applications in the future. However, the open-source nature of the platform will allow users to also adapt the chip design if desired.

    Weaknesses:

    1. The only concern for the HyDrop platform is that it may not be easy ('plug-and-play') to implement for non-specialist labs. As such, its adoption will likely be linked to some sort of support for such users.
      However, this detracts nothing from the validity of the presented manuscript that will fulfill an urgent need for a high-performing non-commercial platform to make single-cell genomics more equitable.