Core E: Cell sorting, CyTOF and RNA-Seq

Core E description 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 naïve 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).