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Integrated Instrument System for Single Cell Genomics

Leonid S Metelitsa

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National Institutes of Health (NIH)
Although tumors consist of highly heterogeneous mixtures of cells with distinct genetic, phenotypic and functional properties, modern methods of genomic analysis are performed with whole tumor samples, in which biological differences between cells can be nullified by averaging or mistaken for noise. Among the rare and routinely missed cells are cancer stem cells which drive and sustain tumor growth; inflammatory cells such as macrophages which support tumor growth, metastasis and drug resistance; and immune effector cells which have a potential to eradicate the tumor if the mechanism of their suppression is identified. The proposed Integrated Instrument System (IIS) will enable single-cell genomic applications with viable primary patient- derived cells. The IIS combines a Sony SH800 FACS sorter and Fuidigm C1" Single-Cell Auto Prep System. The SH800 and C1" will be placed next to each other on the single platform within the existing flow cytometry core facility at Texas Children's Hospital (TXCH-FCL) and will only be used within the proposed IIS. The most common input cells will be freshly prepared suspensions of viable cells obtained from primary specimens of resected tumors from patients with neuroblastoma, pediatric brain tumors, sarcomas, lymphomas, or patient- specific T cell products for cancer immunotherapy. The SH800 will be used to sort subpopulations of interest based on a combination of known surface markers that can be detected with the available antibodies. A defined number of cells will be directly sorted into the loading well of the FluidigmC1" chip under Baker BioProtect Jr BSL-II biohazard containment cabinet and with the use of the included Aerosol Management System. The C1" chip has an integrated fluidic circuit that captures and process 96 individual cells in parallel. The C1" Single-Cell Auto Prep System will test cell viability and perform cell lysis, DNA/RNA extraction, reverse transcription, and amplification of cDNA from 96 single cells for downstream applications such as sequencing of single-cell libraries or gene-expression analysis with the established platforms (e.g. Illumina or Affymetrics). The system will be fully integrated in the existing clinical and research infrastructure at BCM and provide a missing block for enabling highly efficient processing of viable primary tissue specimens from pediatric cancer patients for single cell genomic applications. The results are expected to dramatically accelerate progress in multiple NIH-funded projects from 12 investigators, studying pediatric solid and hematologic malignancies or patient-specific therapeutic T cell products and lead to the paradigm-changing discoveries in the search for the cure of pediatric cancer.

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