Abstract
Lysine lactylation is a distinctive histone modification that plays a crucial role in epigenetic regulation and gene transcription. Here, we present a protocol for studying the genomic profile of histone lactylation with the CUT&RUN assay in tumor-associated macrophages. We describe steps for preparing live cells, gating strategies for isolating glucose transporter type 1 + (GLUT1+) monocyte-derived macrophages (MDMs) by fluorescence-activated cell sorting (FACS), binding lactyl-specific primary antibodies, and quantifying DNA using qPCR. This protocol is applicable to both tumor-derived and in-vitro-generated bone-marrow-derived macrophages. For complete details on the use and execution of this protocol, please refer to De Leo et al.1.