20002159701
Total RNA was extracted from freshly frozen tumor samples using Qiagen kits (Toronto, ON, Canada). Bulk RNA sequencing was performed to evaluate the expression of the TRPC signature. Prior to library construction, rigorous quality control steps were implemented to ensure RNA purity and integrity. This involved initial assessment of RNA purity using Nanodrop2000 (Thermo Scientific; Waltham, MA, USA), agarose gel electrophoresis for RNA integrity and potential contamination, and reconfirmation of RNA integrity using the Agilent 2100 Bioanalyzer (Santa Clara, California, USA). Subsequently, mRNA was purified and randomly fragmented to initiate cDNA synthesis. For library construction, cDNA fragments of 150-200bp length were purified to a concentration of 1.5ng/l using AMPure XP beads (Beckman Coulter, Beverly, USA). Assessment of library effective concentration and RNA quality were performed using Agilent 2100 Bioanalyzer and Qubit2.0 (Thermo Scientific; Waltham, MA, USA). Finally, the libraries underwent sequencing on the HiSeq platform (Illumina, San Diago, CA, USA) following the manufacturers protocols.