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An Automated Microwell Platform for Large-Scale Single Cell RNA-Seq

View Article: PubMed Central - PubMed

ABSTRACT

Recent developments have enabled rapid, inexpensive RNA sequencing of thousands of individual cells from a single specimen, raising the possibility of unbiased and comprehensive expression profiling from complex tissues. Microwell arrays are a particularly attractive microfluidic platform for single cell analysis due to their scalability, cell capture efficiency, and compatibility with imaging. We report an automated microwell array platform for single cell RNA-Seq with significantly improved performance over previous implementations. We demonstrate cell capture efficiencies of >50%, compatibility with commercially available barcoded mRNA capture beads, and parallel expression profiling from thousands of individual cells. We evaluate the level of cross-contamination in our platform by both tracking fluorescent cell lysate in sealed microwells and with a human-mouse mixed species RNA-Seq experiment. Finally, we apply our system to comprehensively assess heterogeneity in gene expression of patient-derived glioma neurospheres and uncover subpopulations similar to those observed in human glioma tissue.

No MeSH data available.


Related in: MedlinePlus

Characterization of Single Cell RNA-Seq Performance.(A) Histogram of the fraction of molecules uniquely aligned to the human transcriptome for a mixed species analysis including human U87 and murine 3T3 cells. The bimodal distribution peaked near zero and one indicate that our single cell profiles are of high purity. Cell-identifying barcodes with purities that deviate significantly from zero or one are indicative of multiplets (microwells containing two or more cells from both species). (B) Heat map of a cell cycle score for each U87 cell indicating the relative expression of genes associated with each of five cell cycle stages. The heat map shows that there are cells in all five cell cycle stages and cells that appear to be in specific intermediate transition states between stages. (C) Same as (B) but for individual 3T3 cells. (D) Histogram showing the number of uniquely aligned molecules per cell for human U87 cells. (E) Histogram showing the number of genes detected per cell for human U87 cells. (F) Sub-sampling saturation curve for the number of molecules detected per human U87 cell as a function of the number of uniquely aligned reads sampled. (G) Same as (F) for the number of genes detected per human U87 cell. (H–K) Same analysis as in (D–G) for murine 3T3 cells.
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f3: Characterization of Single Cell RNA-Seq Performance.(A) Histogram of the fraction of molecules uniquely aligned to the human transcriptome for a mixed species analysis including human U87 and murine 3T3 cells. The bimodal distribution peaked near zero and one indicate that our single cell profiles are of high purity. Cell-identifying barcodes with purities that deviate significantly from zero or one are indicative of multiplets (microwells containing two or more cells from both species). (B) Heat map of a cell cycle score for each U87 cell indicating the relative expression of genes associated with each of five cell cycle stages. The heat map shows that there are cells in all five cell cycle stages and cells that appear to be in specific intermediate transition states between stages. (C) Same as (B) but for individual 3T3 cells. (D) Histogram showing the number of uniquely aligned molecules per cell for human U87 cells. (E) Histogram showing the number of genes detected per cell for human U87 cells. (F) Sub-sampling saturation curve for the number of molecules detected per human U87 cell as a function of the number of uniquely aligned reads sampled. (G) Same as (F) for the number of genes detected per human U87 cell. (H–K) Same analysis as in (D–G) for murine 3T3 cells.

Mentions: Figure 3A shows a histogram of the fraction of molecules that uniquely aligned to either the human or murine transcriptome but that aligned best to the human transcriptome for each cell. The bimodal distribution indicates that almost all of the molecules detected for roughly half of the cells originate from human mRNA versus murine mRNA for the remaining half. Because the original mixture was comprised of about 50% human and 50% murine cells, this implies that our single cell RNA-Seq profiles are quite pure (median purity of >98.8%). Cell barcodes associated with a significant number of both human and murine transcripts (<90% purity for the species with the most transcripts) likely originate from “multiplets” or instances in which two or more cells of both species were captured in a single microwell (<0.8% of cell barcodes).


An Automated Microwell Platform for Large-Scale Single Cell RNA-Seq
Characterization of Single Cell RNA-Seq Performance.(A) Histogram of the fraction of molecules uniquely aligned to the human transcriptome for a mixed species analysis including human U87 and murine 3T3 cells. The bimodal distribution peaked near zero and one indicate that our single cell profiles are of high purity. Cell-identifying barcodes with purities that deviate significantly from zero or one are indicative of multiplets (microwells containing two or more cells from both species). (B) Heat map of a cell cycle score for each U87 cell indicating the relative expression of genes associated with each of five cell cycle stages. The heat map shows that there are cells in all five cell cycle stages and cells that appear to be in specific intermediate transition states between stages. (C) Same as (B) but for individual 3T3 cells. (D) Histogram showing the number of uniquely aligned molecules per cell for human U87 cells. (E) Histogram showing the number of genes detected per cell for human U87 cells. (F) Sub-sampling saturation curve for the number of molecules detected per human U87 cell as a function of the number of uniquely aligned reads sampled. (G) Same as (F) for the number of genes detected per human U87 cell. (H–K) Same analysis as in (D–G) for murine 3T3 cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5037380&req=5

f3: Characterization of Single Cell RNA-Seq Performance.(A) Histogram of the fraction of molecules uniquely aligned to the human transcriptome for a mixed species analysis including human U87 and murine 3T3 cells. The bimodal distribution peaked near zero and one indicate that our single cell profiles are of high purity. Cell-identifying barcodes with purities that deviate significantly from zero or one are indicative of multiplets (microwells containing two or more cells from both species). (B) Heat map of a cell cycle score for each U87 cell indicating the relative expression of genes associated with each of five cell cycle stages. The heat map shows that there are cells in all five cell cycle stages and cells that appear to be in specific intermediate transition states between stages. (C) Same as (B) but for individual 3T3 cells. (D) Histogram showing the number of uniquely aligned molecules per cell for human U87 cells. (E) Histogram showing the number of genes detected per cell for human U87 cells. (F) Sub-sampling saturation curve for the number of molecules detected per human U87 cell as a function of the number of uniquely aligned reads sampled. (G) Same as (F) for the number of genes detected per human U87 cell. (H–K) Same analysis as in (D–G) for murine 3T3 cells.
Mentions: Figure 3A shows a histogram of the fraction of molecules that uniquely aligned to either the human or murine transcriptome but that aligned best to the human transcriptome for each cell. The bimodal distribution indicates that almost all of the molecules detected for roughly half of the cells originate from human mRNA versus murine mRNA for the remaining half. Because the original mixture was comprised of about 50% human and 50% murine cells, this implies that our single cell RNA-Seq profiles are quite pure (median purity of >98.8%). Cell barcodes associated with a significant number of both human and murine transcripts (<90% purity for the species with the most transcripts) likely originate from “multiplets” or instances in which two or more cells of both species were captured in a single microwell (<0.8% of cell barcodes).

View Article: PubMed Central - PubMed

ABSTRACT

Recent developments have enabled rapid, inexpensive RNA sequencing of thousands of individual cells from a single specimen, raising the possibility of unbiased and comprehensive expression profiling from complex tissues. Microwell arrays are a particularly attractive microfluidic platform for single cell analysis due to their scalability, cell capture efficiency, and compatibility with imaging. We report an automated microwell array platform for single cell RNA-Seq with significantly improved performance over previous implementations. We demonstrate cell capture efficiencies of &gt;50%, compatibility with commercially available barcoded mRNA capture beads, and parallel expression profiling from thousands of individual cells. We evaluate the level of cross-contamination in our platform by both tracking fluorescent cell lysate in sealed microwells and with a human-mouse mixed species RNA-Seq experiment. Finally, we apply our system to comprehensively assess heterogeneity in gene expression of patient-derived glioma neurospheres and uncover subpopulations similar to those observed in human glioma tissue.

No MeSH data available.


Related in: MedlinePlus