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Scalable microfluidics for single-cell RNA printing and sequencing.

Bose S, Wan Z, Carr A, Rizvi AH, Vieira G, Pe'er D, Sims PA - Genome Biol. (2015)

Bottom Line: We then develop a scalable technology for genome-wide, single-cell RNA-Seq.Our device generates pooled libraries from hundreds of individual cells with consumable costs of $0.10-$0.20 per cell and includes five lanes for simultaneous experiments.We anticipate that this system will serve as a general platform for single-cell imaging and sequencing.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems Biology, Columbia University Medical Center, New York, NY, 10032, USA. sb3438@columbia.edu.

ABSTRACT
Many important biological questions demand single-cell transcriptomics on a large scale. Hence, new tools are urgently needed for efficient, inexpensive manipulation of RNA from individual cells. We report a simple platform for trapping single-cell lysates in sealed, picoliter microwells capable of printing RNA on glass or capturing RNA on beads. We then develop a scalable technology for genome-wide, single-cell RNA-Seq. Our device generates pooled libraries from hundreds of individual cells with consumable costs of $0.10-$0.20 per cell and includes five lanes for simultaneous experiments. We anticipate that this system will serve as a general platform for single-cell imaging and sequencing.

No MeSH data available.


Related in: MedlinePlus

Schematic and fluorescence imaging data for single-cell RNA printing. a Cells are first deposited in the microwell array by gravity. The glass surface opposite the microwell array is covalently functionalized with oligo(dT) primers for mRNA capture (orange line). The device is then rapidly and conformally sealed against a glass surface in the presence of lysis buffer, flipped over, and held in a sealed position using negative pressure. Single-cell lysates (green) become trapped in the sealed microwells, and mRNA hybridizes to the oligo(dT) primers on the glass surface, resulting in single-cell mRNA ‘prints’ (red lines). b An array of single-cell mRNA prints on a glass coverslip generated using the device in Fig. 1a and imaged after on-chip reverse transcription. The double-stranded RNA/DNA hybrids are stained with SYTOX Orange, an intercalator dye and imaged on the glass surface. More than 96 % of the prints result from individual cells. Note that the bright spots in the image that are not registered with the array originate from genomic DNA aggregates that were not fully removed by DNase digestion. c Close-up images of single-cell RNA printing. The left panel is a bright field image of three cells in individual microwells of the array, the middle panel is a fluorescence image of the corresponding RNA prints on the glass surface after reverse transcription and staining with SYTOX Orange, and the right panel is a fluorescence image of the glass surface after RNase digestion, demonstrating that the fluorescent prints originate from captured RNA
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Fig1: Schematic and fluorescence imaging data for single-cell RNA printing. a Cells are first deposited in the microwell array by gravity. The glass surface opposite the microwell array is covalently functionalized with oligo(dT) primers for mRNA capture (orange line). The device is then rapidly and conformally sealed against a glass surface in the presence of lysis buffer, flipped over, and held in a sealed position using negative pressure. Single-cell lysates (green) become trapped in the sealed microwells, and mRNA hybridizes to the oligo(dT) primers on the glass surface, resulting in single-cell mRNA ‘prints’ (red lines). b An array of single-cell mRNA prints on a glass coverslip generated using the device in Fig. 1a and imaged after on-chip reverse transcription. The double-stranded RNA/DNA hybrids are stained with SYTOX Orange, an intercalator dye and imaged on the glass surface. More than 96 % of the prints result from individual cells. Note that the bright spots in the image that are not registered with the array originate from genomic DNA aggregates that were not fully removed by DNase digestion. c Close-up images of single-cell RNA printing. The left panel is a bright field image of three cells in individual microwells of the array, the middle panel is a fluorescence image of the corresponding RNA prints on the glass surface after reverse transcription and staining with SYTOX Orange, and the right panel is a fluorescence image of the glass surface after RNase digestion, demonstrating that the fluorescent prints originate from captured RNA

Mentions: Our microfluidic platform is comprised of a simple flow cell with an array of microwells embedded in either the top or bottom of the device similar to what we have reported previously for high-throughput DNA sequencing [21] and digital PCR [22]. We drive fluids through the flow cell manually at a standard laboratory bench by laminar flow using a syringe or pipette. Fluid exchange in the microwells occurs by diffusion, while cells and beads can be loaded by gravity. We fabricate the microwell arrays in polydimethylsiloxane (PDMS), a silicone rubber commonly used in soft lithography [23]. PDMS allows inexpensive, rapid, and repeatable fabrication from molds produced on silicon in photoresist using standard photolithography [23]. In addition, the material properties of PDMS, including its hydrophobicity and flexibility, facilitate reversible sealing of the microwells against a flat surface using mechanical deformation and negative pressure [21, 24] (Fig. 1a) or introduction of oil [25] by laminar flow (Fig. 2a). Several variations on microwell arrays have been reported previously for gene-specific analysis in individual cells [26], targeted analysis of gene panels [27], or paired chain analysis of the antibody repertoire [28]. Here, we have advanced this technology for genome-wide RNA capture and sequencing.Fig. 1


Scalable microfluidics for single-cell RNA printing and sequencing.

Bose S, Wan Z, Carr A, Rizvi AH, Vieira G, Pe'er D, Sims PA - Genome Biol. (2015)

Schematic and fluorescence imaging data for single-cell RNA printing. a Cells are first deposited in the microwell array by gravity. The glass surface opposite the microwell array is covalently functionalized with oligo(dT) primers for mRNA capture (orange line). The device is then rapidly and conformally sealed against a glass surface in the presence of lysis buffer, flipped over, and held in a sealed position using negative pressure. Single-cell lysates (green) become trapped in the sealed microwells, and mRNA hybridizes to the oligo(dT) primers on the glass surface, resulting in single-cell mRNA ‘prints’ (red lines). b An array of single-cell mRNA prints on a glass coverslip generated using the device in Fig. 1a and imaged after on-chip reverse transcription. The double-stranded RNA/DNA hybrids are stained with SYTOX Orange, an intercalator dye and imaged on the glass surface. More than 96 % of the prints result from individual cells. Note that the bright spots in the image that are not registered with the array originate from genomic DNA aggregates that were not fully removed by DNase digestion. c Close-up images of single-cell RNA printing. The left panel is a bright field image of three cells in individual microwells of the array, the middle panel is a fluorescence image of the corresponding RNA prints on the glass surface after reverse transcription and staining with SYTOX Orange, and the right panel is a fluorescence image of the glass surface after RNase digestion, demonstrating that the fluorescent prints originate from captured RNA
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4487847&req=5

Fig1: Schematic and fluorescence imaging data for single-cell RNA printing. a Cells are first deposited in the microwell array by gravity. The glass surface opposite the microwell array is covalently functionalized with oligo(dT) primers for mRNA capture (orange line). The device is then rapidly and conformally sealed against a glass surface in the presence of lysis buffer, flipped over, and held in a sealed position using negative pressure. Single-cell lysates (green) become trapped in the sealed microwells, and mRNA hybridizes to the oligo(dT) primers on the glass surface, resulting in single-cell mRNA ‘prints’ (red lines). b An array of single-cell mRNA prints on a glass coverslip generated using the device in Fig. 1a and imaged after on-chip reverse transcription. The double-stranded RNA/DNA hybrids are stained with SYTOX Orange, an intercalator dye and imaged on the glass surface. More than 96 % of the prints result from individual cells. Note that the bright spots in the image that are not registered with the array originate from genomic DNA aggregates that were not fully removed by DNase digestion. c Close-up images of single-cell RNA printing. The left panel is a bright field image of three cells in individual microwells of the array, the middle panel is a fluorescence image of the corresponding RNA prints on the glass surface after reverse transcription and staining with SYTOX Orange, and the right panel is a fluorescence image of the glass surface after RNase digestion, demonstrating that the fluorescent prints originate from captured RNA
Mentions: Our microfluidic platform is comprised of a simple flow cell with an array of microwells embedded in either the top or bottom of the device similar to what we have reported previously for high-throughput DNA sequencing [21] and digital PCR [22]. We drive fluids through the flow cell manually at a standard laboratory bench by laminar flow using a syringe or pipette. Fluid exchange in the microwells occurs by diffusion, while cells and beads can be loaded by gravity. We fabricate the microwell arrays in polydimethylsiloxane (PDMS), a silicone rubber commonly used in soft lithography [23]. PDMS allows inexpensive, rapid, and repeatable fabrication from molds produced on silicon in photoresist using standard photolithography [23]. In addition, the material properties of PDMS, including its hydrophobicity and flexibility, facilitate reversible sealing of the microwells against a flat surface using mechanical deformation and negative pressure [21, 24] (Fig. 1a) or introduction of oil [25] by laminar flow (Fig. 2a). Several variations on microwell arrays have been reported previously for gene-specific analysis in individual cells [26], targeted analysis of gene panels [27], or paired chain analysis of the antibody repertoire [28]. Here, we have advanced this technology for genome-wide RNA capture and sequencing.Fig. 1

Bottom Line: We then develop a scalable technology for genome-wide, single-cell RNA-Seq.Our device generates pooled libraries from hundreds of individual cells with consumable costs of $0.10-$0.20 per cell and includes five lanes for simultaneous experiments.We anticipate that this system will serve as a general platform for single-cell imaging and sequencing.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems Biology, Columbia University Medical Center, New York, NY, 10032, USA. sb3438@columbia.edu.

ABSTRACT
Many important biological questions demand single-cell transcriptomics on a large scale. Hence, new tools are urgently needed for efficient, inexpensive manipulation of RNA from individual cells. We report a simple platform for trapping single-cell lysates in sealed, picoliter microwells capable of printing RNA on glass or capturing RNA on beads. We then develop a scalable technology for genome-wide, single-cell RNA-Seq. Our device generates pooled libraries from hundreds of individual cells with consumable costs of $0.10-$0.20 per cell and includes five lanes for simultaneous experiments. We anticipate that this system will serve as a general platform for single-cell imaging and sequencing.

No MeSH data available.


Related in: MedlinePlus