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RNA was extracted from FFPE tissue using Amsbio’s ExpressArt FFPE Clear RNA Ready kit (Amsbio LLC, Cambridge, MA). Fragment length was assessed with an RNA 6000 chip on an Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA). RNA-seq libraries were prepared using 20–100 ng total RNA with the Trusight RNA Fusion Panel (Illumina, San Diego, CA), which targets a list of N genes of interest. Each sample was subjected to targeted RNA sequencing on an Illumina MiSeq at 8 samples per flow cell (approximately 3 million reads per sample). All reads were independently aligned with STAR (ver 2.3) and BowTie2 against the human reference genome (hg19) for Manta-Fusion and TopHat-Fusion analysis, respectively, for fusion discovery. The mRNA expression levels of certain genes of interest (BCOR, SS18L1, SSX1, etc.) were evaluated and compared to those of other samples analyzed in the same batch of targeted RNA sequencing platform, including 3 fusion-negative URCS, 2 epithelioid hemangioendotheliomas, one SS with SS18-SSX2 fusion, and one inflammatory myofibroblastic tumor.

RNA was extracted from FFPE tissue using Amsbio’s ExpressArt FFPE Clear RNA Ready kit (Amsbio LLC, Cambridge, MA). The case was tested on the TruSight RNA Fusion Panel (Illumina, San Diego, CA), using 100 ng total RNA for RNA-sequencing libraries preparation. Fragment length was assessed with an RNA 6000 chip on an Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA). The sample was subjected to targeted RNA sequencing on an Illumina MiSeq (~3 million reads per sample). All reads were independently aligned with STAR (version 2.3) and BowTie2 against the human reference genome (hg19) for Manta-Fusion and TopHat-Fusion analysis, respectively. The details of this methodology are as previously described^{4 (link)}.

RNA was extracted from FFPE tissue using Amsbio’s ExpressArt FFPE Clear RNA Ready kit (Amsbio LLC, Cambridge, MA). Fragment length was assessed with an RNA 6000 chip on an Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA). RNA-seq libraries were prepared using 20–100 ng total RNA with the TruSight RNA Fusion Panel (Illumina, San Diego, CA). Each sample was subjected to targeted RNA sequencing on an Illumina MiSeq at 8 samples per flow cell (approximately 3 million reads per sample). All reads were independently aligned with STAR(ver 2.3) and BowTie2 against the human reference genome (hg19) for Manta-Fusion and TopHat-Fusion analysis, respectively. The mRNA expression levels of certain genes of interest (GL1, ACTB1, CBX5, PTCH1, SOX2, VEGF1, CCND1) were evaluated and compared to those of other samples analyzed on the same targeted RNA sequencing platform.