Core E has four functions, serving all 4 projects. First, human and mouse T cells, B cells and monocytes will be sorted from peripheral blood mononuclear cells (PBMCs) and sorted into trizol, RNA extracted, quality controls (bioanalyzer, Agilent TapeStation) performed, libraries prepared and RNA-Seq done to obtain ~50-70 million Illumina reads per sample. Preliminary data show excellent sequencing depth and data quality even in long- term frozen PBMCs. Mouse and human samples are focused on monocyte subsets for project 1, treated human and sorted mouse macrophages for project 2, B1 cells from peritoneal cavity, spleen and bone marrow in wild-type, Cxcr4-/- and Cxcr5-/- mice for project 3, and dextramerhigh, dextramerlow, dextramernegative effector memory (TEM) cells and nave CD4 T cells for project 4. Second, 36 to 42-parameter mass cytometry (CyTOF) followed by high-dimensional analysis (SPADE, viSNE) will be used for unsupervised clustering. We have already acquired or conjugated, titrated and validated more than 100 monoclonal antibodies, developed one human panel that will serve all projects and four human panels that serve each project individually. We will use about 1 million cells from each tube for this, and the remaining about 7 million cells will be used for RNA-Seq. We also will run CyTOF on mouse. CyTOF identifies the known cell types and likely will suggest new, unknown cell types (new subsets of monocytes, B cells, T cells). Third, sorted T cells and antigen presenting cells (APCs) will be distributed to project 4 and sorted monocytes to project 1 for functional experiments in vivo and in vitro as detailed in the projects. Finally, core E will also provide basic bioinformatics support. This includes post-sequencing quality controls for RNA-Seq, mapping reads, differential expression (DE), heat maps, Venn diagrams and principal component analysis (PCA).
Core E narrative For this large effort to be effective and to produce the highest quality data with the best possible quality controls, three key modern technologies are centralized in core E. Millions of immune cells can be sorted in a few minutes in ~16 dimensions using cell surface markers tagged with fluorescent molecules by flow cytometry (FACS). The cells can be sorted in tubes and the messenger ribonucleic acid (mRNA) can be harvested and analyzed by Illumina sequencers. The results give a genome-wide assessment of how much of each gene is transcribed into mRNA, which correlates with the amount of protein made from each gene, an analysis in ~20,000 dimensions (number of genes in genome). The third method uses mass cytometry (CyTOF). Cell surface markers are tagged with rare earth elements (lanthanides), each cell is vaporized and the rare earth elements are detected in a mass spectrometer. The advantage of this method is that each single cell can be interrogated in ~40 dimensions rather than ~16. This is a powerful discovery tool for new immune cell populations. We expect that some immune cell populations will be different between healthy people and people with atherosclerosis (hardening of the arteries).
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