Nencki Institute of Experimental Biology

Microglia accumulate in malignant gliomas and play a pivotal role during early stages of tumor development. Available in vivo single cell RNA studies have probed gene expression in the myeloid cells within glioma tumors only at relatively late stages of the tumor development. Therefore, the changes in gene expression in microglia in response to glioma are not fully characterized. In the current study, using RNA-seq, we characterized the transcriptional response of rat primary microglia in vitro to rat C6 glioma-conditioned medium (GCM), at 3 time-points: 6 h, 24 h, and 48 h; after the change of medium to GCM, as compared to the untreated control (0 h). Each of the 4 investigated groups had 3 biological replicates (performed on different dates, on primary microglia isolated from different rat litters), giving a total of 12 RNA samples. Total RNA was isolated from microglial cultures. RNA libraries were prepared and sequenced on Illumina HiSeq 1500. Raw sequencing data were processed using nextflow nf-core/rnaseq 3.12.0 workflow with the default options. The filtered reads were alligned to GRCr8.114 rat genome using STAR, followed by quantification and mapping to genes using Salmon, and coverage (.bigWig) files were created. The Salmon-generated reads counts file (salmon.merged.gene_counts.tsv) was imported into R environment. Differential expression analysis was performed using DESeq2 1.38.3, for protein coding, miRNA, and lncRNA genes. Additionally, we performed the variance stabilizing transformation (VST), followed by removal of the batch effect of the replicate number using limma 3.54.2. The results are contained in the file GRCr8_newComboDf.tsv.