Protocol to isolate mature thymic T cell subsets using fluorescence-activated cell sorting for assessing gene expression by RNA-seq and transcription factor binding across the genome by CUT&RUN.

Here, we describe steps to isolate mature thymic T cell subsets, namely CD4 single positive (SP), CD8 SP, and invariant natural killer T (iNKT) cells starting from murine total thymocytes using fluorescence-activated cell sorting. We detail protocols to study gene expression by RNA-seq and assess binding of transcription factors across the genome using CUT&RUN. This approach deciphers the molecular principles that govern T cell lineage specification and function. This protocol works well with limited starting material. For complete details on the use and execution of this protocol, please refer to Äijö et al. (2022)..