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. 2014 Sep 26;4:6485.
doi: 10.1038/srep06485.

Co-detection and Sequencing of Genes and Transcripts From the Same Single Cells Facilitated by a Microfluidics Platform

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Free PMC article

Co-detection and Sequencing of Genes and Transcripts From the Same Single Cells Facilitated by a Microfluidics Platform

Lin Han et al. Sci Rep. .
Free PMC article

Abstract

Despite the recent advance of single-cell gene expression analyses, co-measurement of both genomic and transcriptional signatures at the single-cell level has not been realized. However such analysis is necessary in order to accurately delineate how genetic information is transcribed, expressed, and regulated to give rise to an enormously diverse range of cell phenotypes. Here we report on a microfluidics-facilitated approach that allows for controlled separation of cytoplasmic and nuclear contents of a single cell followed by on-chip amplification of genomic DNA and cytoplasmic mRNA. When coupled with off-chip polymerase chain reaction, gel electrophoresis and Sanger sequencing, a panel of genes and transcripts from the same single cell can be co-detected and sequenced. This platform is potentially an enabling tool to permit multiple genomic measurements performed on the same single cells and opens new opportunities to tackle a range of fundamental biology questions including non-genetic cell-to-cell variability, epigenetic regulation, and stem cell fate control. It also helps address clinical challenges such as diagnosing intra-tumor heterogeneity and dissecting complex cellular immune responses.

Figures

Figure 1
Figure 1. Microfluidic device designed for single-cell mRNA/gDNA separation and whole pool amplification.
(a). Photograph showing a PDMS microfluidic device bound on a glass slide. The microfluidics device is filled with food dye for illustration. All flow channels are filled with red dye, and the control channel are filled with green dye. b) Layout of microfluidic device with 7 identical units. Optical photograph of a single unit is overlaid on top. (C). A single K562 cell highlighted by the red square captured in the micro-chamber. (d–f). Schematic depiction of the process flow. (d) A single cell was captured in the microchamber of each unit. (e) The cell membrane was lysed by the membrane-selective lysis buffer. (f) RNA was separated from the intact nucleus. (g) Final products of cDNA and gDNA in separated microchambers are subjected to whole pool amplification.
Figure 2
Figure 2. Selective lysis of cell membrane and extraction of cytoplasmic and nuclear contents respectively.
(a). Summary of 4 representative lysis buffers in terms of their performance for selective cell membrane lysis and subsequent mRNA analysis. (b). Live cell imaging of selective cell lysis in a microfluidic chip. The upper panel shows rapid lysis of cell membrane and release of cytoplasm (pre-stained in green) with no effect on nucleus. The lower panel shows the nucleus remains intact in the lysis buffer for 1 hr and then lysed by a high-pH buffer to release gDNA (pre-stained in red).
Figure 3
Figure 3. PCR detection and sequencing of multiple transcripts in single cells.
(a). PCR detection of transcripts by electrophoretic anlaysis of PCR products from single K562 cell cDNA amplicons and population controls. 14 pairs of primers were used to detect the amplification efficiency of single K562 cell by PCR. R5 and R7 are cDNA amplicons from 2 single K562 cells; cD is cDNA from a population of K562 cells; gD is genomic DNA from a population K562 cells. (b). Sanger sequencing results with the PCR products from the bands corresponding to Creb5, Creb9, CR6, and PMP.
Figure 4
Figure 4. Single-cell mRNA amplicons: quality and purity.
(a). PCR detection of transcripts GAPDH and B2M in 16 single-cell mRNA amplicons. The same amplicons were also checked for the presence of genomic DNA using a primer pair 2p, which detects a sequence in the intron region of chromosome 2. M: molecular weight marker. 1–16: single-cell cDNA amplicons. (b). Next-generation sequencing of three single-cell mRNA amplicons (S1, S2, and S3) and a 1000-cell amplicon (K) showing excellent coverage over the gene sequences in chromosome 1. Blue bars indicate the human genome reference sequence. Non-coding regions are not present in any of these samples. (c). Summary of tag counts for exons and introns. The results are from three single-cell amplicons. The intron tag counts in all these samples are negligible (~0.01%).
Figure 5
Figure 5. PCR detection and sequencing of multiple genes from the gDNA of the same single cells.
(a). a. PCR detection of genes by electrophoretic analysis of PCR products from single K562 cell gDNA amplicons and population controls. 12 pair primers were used to measure the amplification efficiency of single K562 cell gDNA by PCR. D5 and D7 are gDNA amplicons from the same single K562 cells as of R5 and R7 (Fig. 3); gD is genomic DNA of a population of K562 cells. WgD is the gDNA product amplified from a small amount of sample gD used access amplification bias. (b). Sanger sequencing of the PCR products from 2p, 5p, CR6, and PMP bands.

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