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

Dimitris Theofilatos; Tarmo Äijö; Ageliki Tsagaratou

doi:10.1016/j.xpro.2022.101839

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

STAR Protoc. 2022 Nov 11;3(4):101839. doi: 10.1016/j.xpro.2022.101839. eCollection 2022 Dec 16.

Authors

Dimitris Theofilatos¹, Tarmo Äijö², Ageliki Tsagaratou³

Affiliations

¹ Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. Electronic address: [email protected].
² Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
³ Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Microbiology, University of North Carolina, Chapel Hill, NC 27599, USA. Electronic address: [email protected].

Abstract

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.

Keywords: Cell isolation; ChIPseq; Flow Cytometry/Mass Cytometry; Gene Expression; Immunology; RNAseq.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Animals
Flow Cytometry
Gene Expression
Mice
RNA-Seq
T-Lymphocyte Subsets*
Transcription Factors* / genetics

Substances

Transcription Factors

Abstract

Publication types

MeSH terms

Substances

Grants and funding