TempO-Seq Gene Expression Profiling of Intracellular Stained FACS Sorted Cells

PROJECT SUMMARY We propose to develop and validate an intracellular stained Fluorescence Activated Cell Sorter Templated Oligonucleotide Sequencing gene profiling assayicsFACS TempO Seqaddressing an unmet need by enabling low costquantitative gene expression analysis of fixedpermeabilizedand intracellular stained and sorted cellsa method used to purify many different functionally important and rare subsets of cellsincluding single cellson both the FACSAria and benchtop BD FACSMelodySony SHand BioRad S e sortersWe successfully demonstrated feasibility in Phase Ideveloping two protocols for profiling single FACS sorted cellsIn additionwe were able to demonstrate novel observations regarding the stochastic expression of genes at the single cell level due to the performance of the assay and virtual absence of background signal so that trueexpression levels of genes that were off in single cells at the time of fixation could be measuredIn Phase II we will optimize these protocols and validate a menu of assays for human and mousea whole transcriptome assaya surrogate assaya panel comprised of the surrogate assay plus immune response genes for both mouse and humanand a panel comprised of host response genesHIVviral RNA and DNAWe will demonstrate utility to generate time course data of normal T cells activated ex vivo for subsets and single cells within those subsetsFinallywe will generate novel data in the course of demonstrating utility in a mouse model carried out in collaboration with an expert consultantand by data obtained from HIVpatients in atest client arrangement with an expert in HIV researchIn the mouse model we will profile GC B cells undergoing programmed rounds of hypermutation and selectionproliferationand monitor subset purity in a notoriously coalescing surface staining phenotypeIn the human modelthetest client will profile FACS sorted samples from HIVinfected patients undergoing triple antiretroviral therapyARTto characterize the host RNA response and HIVprovirus DNA and viral mRNA harbored within the CDT memory cell compartments and enumerate residual HIVCDT cellsThe assay used will include measurement of viral RNA and DNA at the same time as host genesThe icsFACS TempO Seq protocols and assays developed and validated will address unmet needs in flow cytometry and immunology across a growing range of diseasesincluding infectious diseasesinflammationcancerstrokeand Alzheimer s to name a fewValidation for both the FACSAria and benchtop sorters will address the needs of core labs and their clients and investigators who choose to useor require the specialized single cell performanceof these benchtop sortersIt also lays the groundwork for the development of diagnostic assays on these benchtop sorter platforms using icsFACS TempO Seq PROJECT NARRATIVE We propose to develop and validate protocols and utility of an intracellular stained Fluorescence Activated Cell SortericsFACSTemplated Oligonucleotide SequencingicsFACS TempO Seqplatformincluding human and mouse whole transcriptome assayssurrogate whole transcriptome assaysand combined surrogate immune response gene panelsdemonstrating utility by profiling FACS sorted subsets of Tand Bcells and single cells within those subsets on both the FACSAria and on the BD FACSMelodySony SHand BioRad S e benchtop sortersWe will validate a murine immune response panel and assay and a human infectious disease panel of host response and HIV RNA and DNAand use these to demonstrate utility by providing novel data from profiling subpopulations of murine GC B cells undergoing programmed rounds of hypermutation and selectionproliferationand T cells from HIV infected patients undergoing triple antiretroviral therapyARTto characterize HIVprovirus or viral mRNA and DNA harbored within the CDT memory cell compartments and enumerate residual HIVCDT cellsThese icsFACS TempO Seq assays will be launched commercially and will address an unmet need within the large and growing flow cytometry market and immune response and infectious disease research and therapeutic areas