Nucleotide metabolism in cancer cells fuels a UDP-driven macrophage cross-talk promoting immunosuppression and immunotherapy resistance [RNAseq_Panc02_sgNT_sgCda_tumor]

Purpose: Here we use Here, we used RNA sequencing to identify trascriptictomic profile of sgNT and sgCda PancO2 tumor bearing-mice. Methods: 4x10e6 sgNT and sgCda Panc02 cells were injected subcutaneously (s.c.) in the right flank of the C57BL/6J mouse in a final suspension of 200 µL PBS. Tumor volumes were measured at least three times per week. At day 16 after injection, tumor-bearing mice were sacrificed by cervical dislocation and RNA was isolated from tumor tissue using TRIzol (Life Technologies). Starting from total RNA, poly-adenylated fragments were isolated, reverse transcribed and converted into indexed sequencing libraries using the KAPA stranded mRNA-seq kit (Sopachem, Eke, Belgium). The first 50 bases of these libraries were sequenced on a HiSeq 4000 system (Illumina, San Diego, CA). These raw reads were mapped after removal of the sequencing adapters to the reference transcriptome and genome (GRCm38/mm10) using the Bowtie 2.0 and TopHat 2.0 pipeline (Langmead and Salzberg, 2012). Overall design: Transcriptome profiling was performed in sgNT and sgCda Panc02 tumor murine models. 5x5 design in C57BL/6J mice with tumor tissue RNA sequencing conducted

研究目的：本研究利用RNA测序（RNA sequencing）技术，鉴定携带sgNT与sgCda PancO2肿瘤的小鼠的转录组谱（transcriptomic profile）。
实验方法：将4×10^6个sgNT与sgCda Panc02细胞以200 μL磷酸盐缓冲液（PBS）为终悬液，于C57BL/6J小鼠右侧背部皮下注射。每周至少测量3次肿瘤体积。接种后第16天，采用颈椎脱臼法处死荷瘤小鼠，使用TRIzol（Life Technologies公司）从肿瘤组织中提取总RNA。以总RNA为起始材料，分离聚腺苷酸化RNA片段，经反转录后，使用KAPA链特异性mRNA测序试剂盒（Sopachem，比利时埃克）构建带索引的测序文库。利用HiSeq 4000测序系统（Illumina公司，美国加利福尼亚州圣迭戈）对上述文库的前50个碱基进行测序。去除测序接头后的原始测序读段，使用Bowtie 2.0与TopHat 2.0分析流程（Langmead与Salzberg，2012）比对至参考转录组与参考基因组（GRCm38/mm10）。
实验设计：本研究在sgNT与sgCda PancO2小鼠肿瘤模型中开展转录组测序分析。实验采用5×5实验设计，以C57BL/6J小鼠为对象，对其肿瘤组织进行RNA测序。