Benign Lymph Node
Seven 5 µm sections were prepared at the Winship Cancer Tissue and Pathology Shared Resource Core (CTPSR) for sectioning. One section from each block was stained with Hematoxylin and Eosin (H&E) for histological analysis by a GU pathologist to identify and annotate prostate cancer regions of interest (ROI) within the tissue samples for macrodissection. Annotated H&E slides were aligned to unstained sections and tissue was collected only from the ROI, which was typically >90% tumor. Entire LNM foci were annotated for macrodissection, excluding any benign regions of the lymph nodes. For separate primary tumor foci, independent foci were identified from contralateral aspects of the prostate or separate locations (e.g. apex vs base or anterior vs posterior). The identified regions from all six sections were macrodissected, and the tissue was collected directly into a shearing microtube. Once tissue was obtained, RNA and DNA isolation was performed at the Emory Integrated Genomics Core (EIGC) using the Covaris truXTRAC FFPE total NA kit. Quality control (QC) analysis was also performed by the EIGC on all RNA and DNA extractions, and RNAseq and WES were performed by HudsonAlpha (subunit of Discovery Life Sciences). For RNAseq, we performed 100 bp paired-end sequencing using the SMART-Seq Stranded library prep kit (Takara, 634444).  For WES analysis, libraries were prepared using the KAPA HyperPrep / Covaris Shearing Library Construction, IDT v2 Capture kit and 100 bp PE sequencing was performed on a NovaSeq 6000.