Comparative Transcriptomics of Meso-CAFs and CAF-3

Total RNA of Meso-CAFs and CAF-3 was extracted from primary cell cultures of about 80% confluence with RNeasy Mini Kit (Qiagen Sciences, Germantown, MD, USA) according to the respective protocol of the manufacturer. Whole-genome gene expression analysis was carried out as described previously by Pirker et al. [11 (link)] and Mathieu et al. [13 (link)] using 4x44K whole genome oligonucleotide-based gene expression microarrays (Agilent, Santa Clara, CA, USA) and a G2505B Micro Array Scanner (Agilent, Santa Clara, CA, USA). Gene expression data of Meso-CAFs was compared to CAF-3 and to data of 31 PM cell lines available from a previous publication [11 (link)]. Analysis and comparison of expression data was conducted in R [15 ], and differentially expressed genes between cell types (Meso-CAF, CAF-3, PM) were determined using “limma” package [16 (link)] as previously described by Mohr et al. [17 (link)]. Genes with multiple oligonucleotide probes on the array were summarized to the probe with maximal interquartile range using the package “genefilter” [18 ]. Additional plots were created using GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA). Whole genome gene expression array data from Meso-CAFs and CAF-3 are available at ArrayExpress (https://www.ebi.ac.uk/biostudies/arrayexpress) under the accession number E-MTAB-12177.