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

Abstract

Summary: 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).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• Cell isolation
• Flow Cytometry/Mass Cytometry
• RNAseq
• ChIPseq
• Immunology
• Gene Expression