RNA-seq Analysis of Prostate Cancer Tissues

For RNA-seq analyses, bulk prostate tissues, sorted LIN^-EpCAM⁺ prostate cancer cells and cancer- or spleen-associated CD45⁺CD3⁺CD8⁺ T cells were used. RNA was extracted by using RNAeasy mini kit (Qiagen 74106) or micro kit (Qiagen 74004), and cell line or tumor tissue cDNA library was constructed by using NEBNext® Poly(A) mRNA Magnetic Isolation Module (E7490) and NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (E7530). The cDNA library of CD8⁺ T cell and cancer cell was constructed by using SMART-SEQ 2 protocol. Sequencing was performed by Illumina - HiSeq PE150.
All RNA-seq data were aligned to the mm10 genome using Tophat (version v2.1.1). Differentially expressed genes were identified by Cuffdiff (version v2.2.1)^{64 (link)}. FPKM was used for following analysis and comparison. GSEA analysis was performed as software suggested^{65 (link)}. T cell clonotype diversity analysis was performed by TRUST^{43 (link)}. The pathway activity score was calculated with GSVA⁶⁶ . The expression levels of TLS score genes^{46 (link)} were scaled to the range of 0 to 1, and TLS score was defined as the mean scaled value of related genes for each individual sample,