Modular barcode beads for microfluidic single cell genomics

Delley, Cyrille L.; Abate, Adam R.

doi:10.1101/2020.09.10.292326

Cited by 5 publications

(10 citation statements)

References 44 publications

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“…We here instead immobilized high copy numbers of DNA templates on streptavidin-coated beads. Such beads are used for single-cell sequencing protocols and therefore widely available [42,43]. We showed that in-droplet protein expression from a single DNA-loaded bead is similar to cell-free expression from DNA in solution.…”

Section: Discussionmentioning

confidence: 99%

Efficient multi-gene expression in cell-free droplet microreactors

Sierra

Arold

Grünberg

2021

Preprint

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Cell-free transcription and translation systems promise to accelerate and simplify the engineering of proteins, biological circuits and metabolic pathways. Their encapsulation on microfluidic platforms can generate millions of cell-free reactions in picoliter volume droplets. However, current methods struggle to create DNA diversity between droplets while also reaching sufficient protein expression levels. In particular, efficient multi-gene expression has remained elusive. We here demonstrate that co-encapsulation of DNA-coated beads with a defined cell-free system allows high protein expression while also supporting genetic diversity between individual droplets. We optimize DNA loading on commercially available microbeads through direct binding as well as through the sequential coupling of up to three genes via a solid-phase Golden Gate assembly or BxB1 integrase-based recombineering. Encapsulation with an off-the-shelf microfluidics device allows for single or multiple protein expression from a single DNA-coated bead per 14 pL droplet. We envision that this approach will help to scale up and parallelize the rapid prototyping of more complex biological systems.

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Section: Discussionmentioning

confidence: 99%

Efficient multi-gene expression in cell-free droplet microreactors

Sierra

Arold

Grünberg

2021

Preprint

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“…To generate beads with barcoded primers, acrylamide precursors are combined microfluidically and polymerized in water-in-oil droplets with an acrydited primer. Polyacrylamide beads containing the bound primer are removed from oil using 1H,1H,2H,2H-Perfluoro-1-octanol (Sigma-Aldrich), and barcodes are added using a split-pool ligation-based approach …”

Section: Methodsmentioning

confidence: 99%

“…Cascade Blue is included as a drop dye to enable the merging of all drops as an experimental control. Custom barcode beads are generated as recently described, 25 targeting 16 amplicons of the Mission Bio Acute Myeloid Leukemia panel (Table S1). These amplicons are chosen because K562 and CEM have different single-nucleotide polymorphisms (SNPs) in those genomic locations that allow for their subsequent identification.…”

Section: ■ Experimental Sectionmentioning

confidence: 99%

“…Polyacrylamide beads containing the bound primer are removed from oil using 1H,1H,2H,2H-Perfluoro-1-octanol (Sigma-Aldrich), and barcodes are added using a split-pool ligationbased approach. 25 The droplets containing the cell lysate are prepared on the Mission Bio Tapestri instrument, according to the manufac-turerʼs specifications. Cell lysate droplets are merged with barcoded beads and PCR reagents on a custom microfluidic chip (Figure 4A).…”

Section: ■ Experimental Sectionmentioning

confidence: 99%

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Targeted Single-Cell RNA and DNA Sequencing With Fluorescence-Activated Droplet Merger

et al. 2020

Self Cite

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Analyzing every cell in a diverse sample provides insight into population-level heterogeneity, but abundant cell types dominate the analysis and rarer populations are scarcely represented in the data. To focus on specific cell types, the current paradigm is to physically isolate subsets of interest prior to analysis; however, it remains difficult to isolate and then single-cell sequence such populations because of compounding losses. Here, we describe an alternative approach that selectively merges cells with reagents to achieve enzymatic reactions without having to physically isolate cells. We apply this technique to perform single-cell transcriptome and genome sequencing of specific cell subsets. Our method for analyzing heterogeneous populations obviates the need for pre-or post-enrichment and simplifies single-cell workflows, making it useful for other applications in single-cell biology, combinatorial chemical synthesis, and drug screening.

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“…For the preparation of nuclei for scRNA-seq, we used a modified protocol from the recently published single nuclei preparation toolbox 37 to extract nuclei from snap-frozen mouse cortex samples. Briefly, a ~1 cm 3 frozen piece of mouse cortex tissue was transferred to 0.5 mL of icecold homogenisation buffer (Salt-tris solution -146 mM NaCl, 10 mM Tris 7.5, 1 mM CaCl2, 21 mM MgCl2, 250 mM Sucrose, 0.03% Tween-20, 0.01% BSA, 25 mM KCl, 1 mM 2-Mercaptoethanol, 1X cOmplete protease inhibitor, 0.5U/ul of RNAse In Plus (Promega)) in a Dounce homogenizer mortar and thawed for 2 minutes.…”

Section: Snap-frozen Mouse Cortex Nuclei Extractionmentioning

confidence: 99%

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

Rop

Ismail

González-Blas

et al. 2022

eLife

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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.

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Modular barcode beads for microfluidic single cell genomics

Cited by 5 publications

References 44 publications

Efficient multi-gene expression in cell-free droplet microreactors

Efficient multi-gene expression in cell-free droplet microreactors

Targeted Single-Cell RNA and DNA Sequencing With Fluorescence-Activated Droplet Merger

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

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