HyDrop enables droplet based single-cell ATAC-seq and single-cell RNA-seq using dissolvable hydrogel beads
- VIB-KU Leuven Center for Brain and Disease Research, Belgium
- KU Leuven, Belgium
Abstract
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.
Data availability
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).
Article and author information
Author details
Funding
H2020 European Research Council (724226_cis- CONTROL)
- Stein Aerts
VIB Tech Watch
- Suresh Poovathingal
KU Leuven (C14/18/092)
- Stein Aerts
Fonds Wetenschappelijk Onderzoek (G0B5619N)
- Stein Aerts
Michael J. Fox Foundation for Parkinson's Research (ASAP-000430)
- Christopher Campbell Flerin
Aligning Science Across Parkinson's (ASAP-000430)
- Christopher Campbell Flerin
Foundation Against Cancer (2016-070)
- Stein Aerts
Stichting tegen Kanker
- Jasper Wouters
Belgian Cancer Society
- Jasper Wouters
Fonds Wetenschappelijk Onderzoek
- Florian De Rop
- Carmen Bravo González-Blas
- Jasper Janssens
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
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.
Copyright
© 2022, De Rop et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 10,717
- views
-
- 1,256
- downloads
-
- 55
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
HyDrop enables droplet based single-cell ATAC-seq and single-cell RNA-seq using dissolvable hydrogel beads
eLife 11:e73971.
https://doi.org/10.7554/eLife.73971
Further reading
-
- Chromosomes and Gene Expression
RNA interference (RNAi) is a conserved pathway that utilizes Argonaute proteins and their associated small RNAs to exert gene regulatory function on complementary transcripts. While the majority of germline-expressed RNAi proteins reside in perinuclear germ granules, it is unknown whether and how RNAi pathways are spatially organized in other cell types. Here, we find that the small RNA biogenesis machinery is spatially and temporally organized during Caenorhabditis elegans embryogenesis. Specifically, the RNAi factor, SIMR-1, forms visible concentrates during mid-embryogenesis that contain an RNA-dependent RNA polymerase, a poly-UG polymerase, and the unloaded nuclear Argonaute protein, NRDE-3. Curiously, coincident with the appearance of the SIMR granules, the small RNAs bound to NRDE-3 switch from predominantly CSR-class 22G-RNAs to ERGO-dependent 22G-RNAs. NRDE-3 binds ERGO-dependent 22G-RNAs in the somatic cells of larvae and adults to silence ERGO-target genes; here we further demonstrate that NRDE-3-bound, CSR-class 22G-RNAs repress transcription in oocytes. Thus, our study defines two separable roles for NRDE-3, targeting germline-expressed genes during oogenesis to promote global transcriptional repression, and switching during embryogenesis to repress recently duplicated genes and retrotransposons in somatic cells, highlighting the plasticity of Argonaute proteins and the need for more precise temporal characterization of Argonaute-small RNA interactions.
-
- Chromosomes and Gene Expression
- Genetics and Genomics
A new method for mapping torsion provides insights into the ways that the genome responds to the torsion generated by RNA polymerase II.
