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Vertical flow array chips reliably identify cell types from single-cell mRNA sequencing experiments

View Article: PubMed Central - PubMed

ABSTRACT

Single-cell mRNA sequencing offers an unbiased approach to dissecting cell types as functional units in multicellular tissues. However, highly reliable cell typing based on single-cell gene expression analysis remains challenging because of the lack of methods for efficient sample preparation for high-throughput sequencing and evaluating the statistical reliability of the acquired cell types. Here, we present a highly efficient nucleic reaction chip (a vertical flow array chip (VFAC)) that uses porous materials to reduce measurement noise and improve throughput without a substantial increase in reagent. We also present a probabilistic evaluation method for cell typing depending on the amount of measurement noise. Applying the VFACs to 2580 monocytes provides 1967 single-cell expressions for 47 genes, including low-expression genes such as transcription factors. The statistical method can distinguish two cell types with probabilistic quality values, with the measurement noise level being considered for the first time. This approach enables the identification of various sub-types of cells in tissues and provides a foundation for subsequent analyses.

No MeSH data available.


Schematic structure of the vertical flow array chip and flow-cell device.(a) The vertical flow chips contain 100 microchambers with beads providing porous reaction fields. Each microchamber features a different cell-id tag sequence in the DNA probes immobilized on the beads. (b) Fluorescence microscopy image of the VFAC with trapped cells, where small white dots indicate individual cells. (c) Structure of DNA probes on the beads for mRNA trapping and 1st cDNA synthesis. (d) Schematic diagram of 2nd strand synthesis on beads. (e) Workflow of sample preparation for gene expression analysis using VFACs. Second-strand synthesis and subsequent steps were performed in a tube containing the VFAC.
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f1: Schematic structure of the vertical flow array chip and flow-cell device.(a) The vertical flow chips contain 100 microchambers with beads providing porous reaction fields. Each microchamber features a different cell-id tag sequence in the DNA probes immobilized on the beads. (b) Fluorescence microscopy image of the VFAC with trapped cells, where small white dots indicate individual cells. (c) Structure of DNA probes on the beads for mRNA trapping and 1st cDNA synthesis. (d) Schematic diagram of 2nd strand synthesis on beads. (e) Workflow of sample preparation for gene expression analysis using VFACs. Second-strand synthesis and subsequent steps were performed in a tube containing the VFAC.

Mentions: Our flow-cell device for single-cell analysis is composed of multiple VFACs to enable inexpensive, digital gene expression profiling for thousands of single cells across an arbitrary number of genes without using robotics. Figure 1 presents a schematic of the flow-cell device and the VFACs. Each chip in the device contains 100 vertical flow-through microchambers for synthesizing a cDNA library and a through-hole to capture single cells. After the single cells are drawn and captured in the holes, which are 3–5 micrometers in diameter, the drawing of lysis buffer into the holes enables the extraction of mRNA strands, which are then trapped by DNA probes with poly-T sequences immobilized on the packed beads in the microchambers. Then, 5 × 109 copies of the DNA probes in each chamber (2 orders of magnitude greater than in previous works11) with cell-ID tags are immobilized on the 1 × 105 beads composing the porous structure in each microchamber. This structure enables the highly efficient trapping of mRNA within a few minutes and highly efficient cDNA synthesis from the small amount of mRNA in single cells. The cDNA library from single cells on the beads in the VFACs is then used to construct a sequencing library for high-throughput sequencing by PCR amplification. Gene-specific probes combined with primers are used for PCR (Methods) to acquire sequencing data, and the data are sorted by the cell tag sequences to identify their original positions on the chip. Single-cell gene expression data were acquired from the cells suspended in solution buffer. The high-density microchambers on the chips and the vertical flow system reduced the volume of expensive enzymatic reagent per cell by a factor of 2028, thus reducing the cost of sample preparation compared to reactions performed in tubes. The microchambers on the VFACs have no walls to prevent the exchange of reagents, simplifying the structure of the VFACs and the flow-cell device, and the high-density integration of the microchambers further reduces reagent costs. The crosstalk between wells was suppressed by continuous down flow through the micro-wells.


Vertical flow array chips reliably identify cell types from single-cell mRNA sequencing experiments
Schematic structure of the vertical flow array chip and flow-cell device.(a) The vertical flow chips contain 100 microchambers with beads providing porous reaction fields. Each microchamber features a different cell-id tag sequence in the DNA probes immobilized on the beads. (b) Fluorescence microscopy image of the VFAC with trapped cells, where small white dots indicate individual cells. (c) Structure of DNA probes on the beads for mRNA trapping and 1st cDNA synthesis. (d) Schematic diagram of 2nd strand synthesis on beads. (e) Workflow of sample preparation for gene expression analysis using VFACs. Second-strand synthesis and subsequent steps were performed in a tube containing the VFAC.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5120284&req=5

f1: Schematic structure of the vertical flow array chip and flow-cell device.(a) The vertical flow chips contain 100 microchambers with beads providing porous reaction fields. Each microchamber features a different cell-id tag sequence in the DNA probes immobilized on the beads. (b) Fluorescence microscopy image of the VFAC with trapped cells, where small white dots indicate individual cells. (c) Structure of DNA probes on the beads for mRNA trapping and 1st cDNA synthesis. (d) Schematic diagram of 2nd strand synthesis on beads. (e) Workflow of sample preparation for gene expression analysis using VFACs. Second-strand synthesis and subsequent steps were performed in a tube containing the VFAC.
Mentions: Our flow-cell device for single-cell analysis is composed of multiple VFACs to enable inexpensive, digital gene expression profiling for thousands of single cells across an arbitrary number of genes without using robotics. Figure 1 presents a schematic of the flow-cell device and the VFACs. Each chip in the device contains 100 vertical flow-through microchambers for synthesizing a cDNA library and a through-hole to capture single cells. After the single cells are drawn and captured in the holes, which are 3–5 micrometers in diameter, the drawing of lysis buffer into the holes enables the extraction of mRNA strands, which are then trapped by DNA probes with poly-T sequences immobilized on the packed beads in the microchambers. Then, 5 × 109 copies of the DNA probes in each chamber (2 orders of magnitude greater than in previous works11) with cell-ID tags are immobilized on the 1 × 105 beads composing the porous structure in each microchamber. This structure enables the highly efficient trapping of mRNA within a few minutes and highly efficient cDNA synthesis from the small amount of mRNA in single cells. The cDNA library from single cells on the beads in the VFACs is then used to construct a sequencing library for high-throughput sequencing by PCR amplification. Gene-specific probes combined with primers are used for PCR (Methods) to acquire sequencing data, and the data are sorted by the cell tag sequences to identify their original positions on the chip. Single-cell gene expression data were acquired from the cells suspended in solution buffer. The high-density microchambers on the chips and the vertical flow system reduced the volume of expensive enzymatic reagent per cell by a factor of 2028, thus reducing the cost of sample preparation compared to reactions performed in tubes. The microchambers on the VFACs have no walls to prevent the exchange of reagents, simplifying the structure of the VFACs and the flow-cell device, and the high-density integration of the microchambers further reduces reagent costs. The crosstalk between wells was suppressed by continuous down flow through the micro-wells.

View Article: PubMed Central - PubMed

ABSTRACT

Single-cell mRNA sequencing offers an unbiased approach to dissecting cell types as functional units in multicellular tissues. However, highly reliable cell typing based on single-cell gene expression analysis remains challenging because of the lack of methods for efficient sample preparation for high-throughput sequencing and evaluating the statistical reliability of the acquired cell types. Here, we present a highly efficient nucleic reaction chip (a vertical flow array chip (VFAC)) that uses porous materials to reduce measurement noise and improve throughput without a substantial increase in reagent. We also present a probabilistic evaluation method for cell typing depending on the amount of measurement noise. Applying the VFACs to 2580 monocytes provides 1967 single-cell expressions for 47 genes, including low-expression genes such as transcription factors. The statistical method can distinguish two cell types with probabilistic quality values, with the measurement noise level being considered for the first time. This approach enables the identification of various sub-types of cells in tissues and provides a foundation for subsequent analyses.

No MeSH data available.