Fully integrated single cell imaging and RNA-Seq library preparation

Project SummaryAmong the commercially available systems for single cell isolation and next generation sequencingNGSsample preparationnone are capable of automating both imaging and NGS sample preparation at high throughputWhile single cell genomic analysis has improved in sensitivity and throughputconcomitant improvements in detailed phenotypic characterization of cells has not been integrated into automated workflowsnor have they been scaled beyond a few dozen cells per runCurrentlythe only means of biomarkerbased sorting of cells prior to single cell sequencing is either fluorescence activated cell sortingFACSor imaging cells on a microscope which is separate from an automated sample preparation instrumente gFluidigm CWaferGen ICELLFor exampleX Genomicstechnology relies on upfront FACS purification of cell populationsand still cannot connect these relatively superficial phenotypic observations to downstream NGS dataFluidigm s Csystem allows imaging of the approximatelycells loaded on a microfluidic device on a separate microscopebut this method results in significant rates of multiplets and requires investigators to construct their own imaging methodsequipment and softwareindependent of the CsystemFluidigm s Polaris allows both imaging and NGS sample preparation in one instrument but is limited in throughput tocells per run and represents a significant capital expenseTo address the unmet need for integrated imaging and NGS sample preparation in a singlehigh throughputcost effective systemCell Microsystems proposes here the development of the AIR FLOWSystemUsing our core CellRaft Technologythe AIR FLOWwill allow multi channel fluorescent imagingisolation of single cells and RNA Seq library preparationThe AIR FLOWSystem will allow multi channel automated imaging of surface markersmorphology and even subcellular featureswhile integrating a microfluidic sample preparation method developed by Peter SimsPhD of Columbia University to construct next generation sequencingNGStranscriptomic librariesDrSimssample preparation technology is highly complementary with the CellRaft technologyboth rely on microwell arraysfluorescence imaging and the same biocompatible materialsThis approach employs optically barcoded beads for mRNA capture and sample preparationallowing the resulting sequencing data to be directly linked to imaging data on a cell to cell basisAlsoby integrating these two technologiesthroughputs of several thousand cells perhr run will be easily achievableBased on our Phase I dataCell Microsystemscore CellRaft Technologyprovides key advantages over existing microfluidic technologieseffectively eliminating cell to cell cross contaminationreducing sample input requirements and providing a less stressful environment which reduces transcriptomic artifactsDuring this Phase II programwe will integrate DrSimstechnology with the core CellRaft Technology in the AIR FLOWSystem Project NarrativeSingle cell sequencing is often accomplished by fluorescence based sorting of cells to purify a population of interestfollowed by dropletor microfluidics based preparation of a sequencing libraryAfter sequencing howeverphenotypic imaging and fluorescent marker data is left unconnected to genomic and or transcriptomic analysis on a cell to cell basisA new systemthe Cell MicrosystemsAIR FLOWis proposed hereand will allow imaging based sorting of single cells followed by integrated microfluidic transcriptomic sample preparationAmong the commercially available optionsno single instrument is capable of this fully integrated workflow