Protocol to analyze immune cells in the tumor microenvironment by transcriptome using machine learning

Immunotherapy is a promising strategy to treat cancer. Here, we present a protocol for analyzing the transcriptome-based phenotypic alterations and immune cell infiltration in the tumor microenvironment. We describe steps for integrating single-cell RNA sequencing (scRNA-seq) data, comparing phenotypes and origins of mononuclear phagocytes, inferring the differentiation trajectory and infiltration process, and identifying infiltration-associated genes using machine learning. We then detail procedures for exploring the impact of these genes in prognosis through the integrated microarray and bulk RNA-seq data to obtain potential drug targets. For complete details on the use and execution of this protocol, please refer to Liao et al.1 [Display omitted] •Integrate and analyze the scRNA-seq data•Obtain genes from cell trajectories•Train GBDT models to find phenotype-associated genes•Perform analysis combined with bulk RNA-seq data to find potential drug targets Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Immunotherapy is a promising strategy to treat cancer. Here, we present a protocol for analyzing the transcriptome-based phenotypic alterations and immune cell infiltration in the tumor microenvironment. We describe steps for integrating single-cell RNA sequencing (scRNA-seq) data, comparing phenotypes and origins of mononuclear phagocytes, inferring the differentiation trajectory and infiltration process, and identifying infiltration-associated genes using machine learning. We then detail procedures for exploring the impact of these genes in prognosis through the integrated microarray and bulk RNA-seq data to obtain potential drug targets.