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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

Flow cell device for single-cell RNA-Seq. a Graphical representation of our five-lane microwell array flow cell device for single-cell RNA-Seq. b Schematic of on-chip steps for single-cell RNA-Seq. After depositing cells, barcoded capture beads (barcode sequences represented as different colors), and sealing as in Fig. 2a, single-cell lysates (green) are trapped in individual microwells and mRNA hybridizes to the barcoded capture beads. The device is unsealed and rapidly washed by flow before on-chip, solid-phase reverse transcription and second-strand synthesis followed by elution and pre-amplification of the pooled library by in vitro transcription. c Montage of fluorescence images from part of one lane of the device in (a) showing beads (red) and cells (blue) loaded in the array. Note that this image was acquired following cell lysis while the device is sealed, and so the blue live stain fills the entire volume of the corresponding microwell and is confined to the microwell by sealing
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Fig4: Flow cell device for single-cell RNA-Seq. a Graphical representation of our five-lane microwell array flow cell device for single-cell RNA-Seq. b Schematic of on-chip steps for single-cell RNA-Seq. After depositing cells, barcoded capture beads (barcode sequences represented as different colors), and sealing as in Fig. 2a, single-cell lysates (green) are trapped in individual microwells and mRNA hybridizes to the barcoded capture beads. The device is unsealed and rapidly washed by flow before on-chip, solid-phase reverse transcription and second-strand synthesis followed by elution and pre-amplification of the pooled library by in vitro transcription. c Montage of fluorescence images from part of one lane of the device in (a) showing beads (red) and cells (blue) loaded in the array. Note that this image was acquired following cell lysis while the device is sealed, and so the blue live stain fills the entire volume of the corresponding microwell and is confined to the microwell by sealing

Mentions: We constructed a PDMS microwell device containing five flow channel lanes for physical multiplexing of samples and >10,000 microwells (Fig. 4a). The cylindrical microwells are 50 μm in diameter and height with a volume of <100 pL. Cells are loaded in individual microwells randomly, according to Poisson statistics, such that the majority of cell-containing wells contain one cell. We tune the concentration of our cellular suspension to avoid overloading the microwell array. Specifically, if we capture approximately 100 cells in every 1,000 microwells of a given array, then <5 % of microwells will contain more than one cell. We then load beads into the wells at a somewhat higher density because the mean diameter of the beads (approximately 30 μm) significantly reduces the probability double-loading (Fig. 4bc). While we occasionally observe microwells with more than one bead or more than one cell, size constraints make it rare to observe both beads and cells in an overloaded microwell. Given our pool of 960 cell-identifying barcodes and five lanes, the capacity of this system for single-cell RNA-Seq is approximately 600 cells at a unique barcoding rate of >94 %. We can scale our system and increase capacity simply by synthesizing additional barcodes and/or adding microwells to our device.Fig. 4


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)

Flow cell device for single-cell RNA-Seq. a Graphical representation of our five-lane microwell array flow cell device for single-cell RNA-Seq. b Schematic of on-chip steps for single-cell RNA-Seq. After depositing cells, barcoded capture beads (barcode sequences represented as different colors), and sealing as in Fig. 2a, single-cell lysates (green) are trapped in individual microwells and mRNA hybridizes to the barcoded capture beads. The device is unsealed and rapidly washed by flow before on-chip, solid-phase reverse transcription and second-strand synthesis followed by elution and pre-amplification of the pooled library by in vitro transcription. c Montage of fluorescence images from part of one lane of the device in (a) showing beads (red) and cells (blue) loaded in the array. Note that this image was acquired following cell lysis while the device is sealed, and so the blue live stain fills the entire volume of the corresponding microwell and is confined to the microwell by sealing
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: Flow cell device for single-cell RNA-Seq. a Graphical representation of our five-lane microwell array flow cell device for single-cell RNA-Seq. b Schematic of on-chip steps for single-cell RNA-Seq. After depositing cells, barcoded capture beads (barcode sequences represented as different colors), and sealing as in Fig. 2a, single-cell lysates (green) are trapped in individual microwells and mRNA hybridizes to the barcoded capture beads. The device is unsealed and rapidly washed by flow before on-chip, solid-phase reverse transcription and second-strand synthesis followed by elution and pre-amplification of the pooled library by in vitro transcription. c Montage of fluorescence images from part of one lane of the device in (a) showing beads (red) and cells (blue) loaded in the array. Note that this image was acquired following cell lysis while the device is sealed, and so the blue live stain fills the entire volume of the corresponding microwell and is confined to the microwell by sealing
Mentions: We constructed a PDMS microwell device containing five flow channel lanes for physical multiplexing of samples and >10,000 microwells (Fig. 4a). The cylindrical microwells are 50 μm in diameter and height with a volume of <100 pL. Cells are loaded in individual microwells randomly, according to Poisson statistics, such that the majority of cell-containing wells contain one cell. We tune the concentration of our cellular suspension to avoid overloading the microwell array. Specifically, if we capture approximately 100 cells in every 1,000 microwells of a given array, then <5 % of microwells will contain more than one cell. We then load beads into the wells at a somewhat higher density because the mean diameter of the beads (approximately 30 μm) significantly reduces the probability double-loading (Fig. 4bc). While we occasionally observe microwells with more than one bead or more than one cell, size constraints make it rare to observe both beads and cells in an overloaded microwell. Given our pool of 960 cell-identifying barcodes and five lanes, the capacity of this system for single-cell RNA-Seq is approximately 600 cells at a unique barcoding rate of >94 %. We can scale our system and increase capacity simply by synthesizing additional barcodes and/or adding microwells to our device.Fig. 4

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