Comment[GEAAccession] E-GEAD-394 MAGE-TAB Version 1.1 Investigation Title Comprehensive analysis of the effects of damage-associated molecules derived from a canine tumor cell on macrophages Experiment Description Dying or damaged cells release their cellular components extracellularly if they are not eradicated by the immune system appropriately. Since aberrant exposure of damage-associated molecules to the immune system is often linked to the pathogenesis of inflammation and cancer, elucidating which and how sterile immune responses are induced by damage-associated molecules is of great importance. In this study, we show that prostaglandin E2 (PGE2) is produced in necrotic supernatants from several types of canine tumor cell lines. Inhibition of PGE2 production by treatment of indomethacin, a potent inhibitor for cyclooxygenase enzymes, enhances the induction of Tnf mRNA expression in RAW264.7 cells by necrotic supernatants. These results prompted us to investigate which genes are induced by damage-associated molecules by comprehensive gene expression analysis. We performed RNA-sequencing analysis by using total RNA from RAW264.7 cells treated with necrotic supernatant derived from Sora cells, which is canine urothelial carcinoma cell lines, in combination with indomethacin treatment. Experimental Design stimulus or stress design Experimental Factor Name stimulus condition Experimental Factor Type stimulus condition Person Last Name eto Person First Name shotaro Person Affiliation Research Center for Advanced Science and Technology (RCAST), The University of Tokyo Person Roles submitter Public Release Date 2020-09-08 Protocol Name P-GEAD-631 P-GEAD-632 P-GEAD-633 P-GEAD-634 P-GEAD-635 Protocol Type sample collection protocol nucleic acid extraction protocol nucleic acid library construction protocol nucleic acid sequencing protocol normalization data transformation protocol Protocol Description RAW264.7 cells were stimulated with a necrotic supernatant from Sora cells pre-treated by indomethacin (Indo) or DMSO (Mock) for 4 hours. PBS was used as a control instead of the supernatant. Total RNA was extracted from RAW264.7 cells using NucleoSpin RNA II (MACHEREY NAGEL). Poly(A) mRNA was enriched using magnet beads-conjugated oligo(dT) and following library preparation for high-throughput sequencing was conducted according to the manufacturers procedure (NEBNext Ultra II RNA Library Prep Kit for Illumina; New England BioLabs). Approximately 250 ng of total RNA was used as initial input for mRNA selection and adapter-ligated double stranded cDNA fragment was amplified 12 cycles PCR, which incorporates Illumina P5/P7 adapters and sample-specific barcode sequence. Fragment size and quantity of libraries established were confirmed by Qubit DNA Assay (Invitrogen) and DNA SceenTape (TapeStation; Agilent Technologies). Libraries with unique sample barcodes were pooled together and loaded onto an Illumina HiSeq/NovaSeq instrument according to manufacturers instructions (Illumina). Sequencing was carried out using 150 bp paired-end (PE) configuration. Image analysis, base calling and demultiplex were conducted by the Illumina standard software. Approximately 20M PE reads (6 Gb output in 150 bp PE configuration) per samples were obtained. The raw sequencing reads were filtered to remove adapter and low quality reads. The resulting clean reads were used for mapping to the reference genome (Mus musculus; Ensembl/GRCm38), quantifying gene expression, studying differential gene expression and further downstream analyses. Hisat2 (v2.0.1) was used to index reference genome sequence. Finally, clean data were aligned to reference genome via software Hisat2 (v2.0.1). For expression analysis, transcripts in fasta format are converted from known gff annotation file and indexed properly. Then, with the file as a reference gene file, HTSeq (v0.6.1) estimated gene and isoform expression levels from the pair-end clean data. SDRF File E-GEAD-394.sdrf.txt Comment[AEExperimentType] RNA-seq of coding RNA Comment[BioProject] PRJDB10461 Comment[Last Update Date] 2020-09-08