Transcriptomic Analysis of TAMs in DMBA-Treated Mice

CD45⁺ CD11b⁺ F4/80⁺ TAMs were sorted using SH800S sorter (Sony). Cell lysates were prepared in 5 μL buffer TCL (catalog no. 1031576, Qiagen) + 1% β-mercaptoethanol (catalog no. 21985-023, Thermo Fisher Scientific). Samples were sent to Broad Genomic Service, and the SmartSeq2 platform was used to generate RNA-Seq data. Differentially expressed genes were determined using Limma package (51 (link)) in R. GSEA was performed using GSEA software and plotted by ClusterProfiler (52 (link)) and ggplot2 packages in R. RNA-Seq data were analyzed for gene set enrichment in DMBA3-4-TAMs compared with DMSO3-1-TAMs. Original data are available as the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus database (GSE237536).

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Huang M., Xia Y., Li K., Shao F., Feng Z., Li T., Azin M, & Demehri S. (2023). Carcinogen exposure enhances cancer immunogenicity by blocking the development of an immunosuppressive tumor microenvironment. The Journal of Clinical Investigation, 133(20), e166494.

Publication 2023

SH800S sorter β-mercaptoethanol

Buffer Cd11b Cell Gene Gene expression Genomic Mercaptoethanol Rna seq Tams

Corresponding Organization :

Other organizations : Center for Cancer Research, Harvard University, Massachusetts General Hospital, University of Science and Technology of China

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Variable analysis

independent variables

Treatment with DMBA3-4 vs. DMSO3-1

dependent variables

Gene expression profiles of TAMs

control variables

Cell sorting method (CD45+, CD11b+, F4/80+ TAMs)
Cell lysis protocol (TCL buffer with β-mercaptoethanol)
RNA-Seq platform (SmartSeq2)
Differential expression analysis (Limma package in R)
Gene set enrichment analysis (GSEA software, ClusterProfiler and ggplot2 packages in R)

controls

Positive control: Not specified
Negative control: DMSO3-1-TAMs

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