Technology Core

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PROJECT SUMMARY/ABSTRACT – Technology Core This innovative Systems Biology Center application seeks to model the complex host/pathogen interactions occurring in patients with severe pneumonia. The overall goals of the Technology Core (TechCore) are to provide sample processing, biobanking, and data generation support for all Projects and other Cores in the Super- Successful Clinical Response In Pneumonia Therapy (SCRIPT2) Systems Biology Center. Our Technology Core is uniquely poised to contribute to the success of the SCRIPT2 Study, as we possess expertise in flow cytometry, cell sorting, cryopreservation, and various next-generation sequencing (NGS) techniques, including bulk and single-cell technologies for gene expression (single-cell RNA-seq) and protein expression profiling (cellular indexing of transcriptomes and epitopes [CITE]-seq), T and B cell receptor (TCR and BCR) clonotyping, genome- wide DNA methylation analysis, metagenomics sequencing, and deep pathogen sequencing. The Technology Core will process clinical samples, cryopreserve them, and then cryorecover them for pre-designated NGS assays in close coordination with the Projects and other Cores. The data generated from the TechCore’s activities will feed back to the DMBI and Modeling Cores to support the aims of Projects 1 and 2 as well as iteratively inform sample selection to determine the most informative NGS assays to perform on a given sample. The Technology Core has conceptualized its activities into two Specific Aims: Aim 1. To immunophenotype and perform biobanking of BAL, nasal curettage, and blood samples obtained from patients with severe pneumonia for subsequent NGS analysis. In Aim 1.1, we will use multiparameter flow cytometry to immunophenotype subsets of epithelial and immune cells from samples obtained from patients with severe pneumonia. In Aim 1.2, we will perform cryopreservation of all samples using techniques that allow for downstream NGS assays on selected samples. Aim 2. To perform NGS assays, including single-cell RNA-seq, CITE-seq, single-cell TCR and BCR clonotyping, T cell subset DNA methylation analysis, metagenomics sequencing, and deep pathogen sequencing on selected cryopreserved samples. We will cryorecover samples selected by the Projects in conjunction with the DMBI and Modeling Cores to perform pre-designated NGS assays and subsequent bioinformatics processing. We will also perform other assays, such as cytokine profiling, as directed by the Projects.