Tissue Sampling and RNA Sequencing Protocol

Fresh-frozen tissue samples were partially thawed in 1% formaldehyde/PBS (Phosphate-buffered saline) and continued to fix for 10 min at room temperature. Fixed tissue was rinsed in PBS and embedded in OCT (optimal cutting temperature) compound (4583 Tissue-Tek, Sakura Finetek, Tokyo, Japan). Tissue samples were sectioned (10 μm) and placed on membrane slides (Pen-membrane, Leica, Tokyo Japan). Sections were stained with hematoxylin and dehydrated by ethanol. Tissue parts were obtained by LMD (LMD6000, Leica) [12 (link)]. For the isolation of total RNA, sections were processed with an RNAeasy FFPE Kit (73504, Qiagen, Hilden, Germany). RNA yield and degradation status were monitored by a Bioanalyzer RNA Pico Kit (5067–1513, Agilent Technologies, Santa Clara, CA, USA). Total RNA samples (quantities in ng; Normal: range = 10.5–64.7, mean = 33.6; Tumor: range = 2.2–98.2, mean = 42.7; Stroma: range = 4.2–110, mean = 28.5) were converted into sequencing libraries using a SMARTer Stranded Total RNA Sample Prep Kit (635005, Takara Bio, Shiga, Japan). Sequencing was performed with Illumina NextSeq500 (Illumina, San Diego, CA, USA) to obtain 2 × 36-base reads.