RNA sequencing and analysis
tumor, tumors
TUMOR, TUMORS
MeVa2.1.dOVA tumor–bearing mice were sacrificed after 13 d of treatment and tumors harvested and snap-frozen for RNA sequencing. RNA extraction and sequencing was performed by CeGat. RNA was isolated from samples using RNeasy Mini kit (Qiagen) as per the manufacturer’s instructions. Quality and quantity of the total RNA was assessed by Qubit (Thermo Fisher Scientific) and Bioanalyzer (Agilent). Total RNA samples having RIN >8 were subjected to library generation. Strand-specific libraries were generated using the TruSeq Stranded mRNA sample preparation kit (Illumina Inc.) according to the manufacturer's instructions. The libraries were sequenced with NovaSeq 6000. Demultiplexing of the sequencing reads was performed with Illumina bcl2fastq (2.20). Adapters were trimmed with Skewer (v 0.2.2). The quality of the FASTQ files was analyzed with FastQC (v 0.11.5-cegat). The samples were mapped with STAR (v 2.7.3a) to mouse reference genome Mus_musulus.GRCm39.105 using default settings. The read counts were computed with HTseq-count (v 0.12.4) and were analyzed with DESeq2 (v 1.30.1). Centering of the normalized gene expression read counts was performed by subtracting the row means and scaling by dividing the columns by the SD to generate a z-score. Gene set enrichment analysis was performed using GSEA (v 4.2.3, Broad Institute).