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Summary Single cell RNA sequencing of human thymic cells is dependent on isolation of highly pure and viable cell populations. This protocol describes the isolation of CD34+ progenitor and more differentiated CD34– fractions from post-natal thymic tissue to study thymopoiesis. CD34+ cells represent, Graphical Abstract, Highlights • Protocol for processing of human thymus for single cell RNA-seq • Thymus dissection and density gradient centrifugation isolate mononuclear cells • Magnetic-activated cell separation isolates CD34+/CD34– cells • Fluorescence-activated cell separation isolates populations for RNA-seq, Single cell RNA sequencing of human thymic cells is dependent on isolation of highly pure and viable cell populations. This protocol describes the isolation of CD34+ progenitor and more differentiated CD34– fractions from post-natal thymic tissue to study thymopoiesis. CD34+ cells represent