Differential expression of murine syngeneic orthotopic glioblastoma tumour models

Bulk RNA sequencing analysis of murine syngeneic orthotopic GL261-luc2 and CT2A-luc glioblastoma tumour models Overall design: GL261-luc2, CT2A-luc, GL261-hCD19-luc2, or CT2A-hCD19-luc cells were implanted into murine brains. GL261-luc2 and CT2A-luc tumors were extracted from mouse brains and total RNA was extracted using RNeasy Plus Mini kit and sequencing libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina. mRNAs were enriched with Oligod(T) beads and fragmented for 15 minutes at 94 °C. First-strand and second-strand cDNA were synthesized, end repaired, adenylated at 3'ends, and ligated to universal adapters; followed by index addition and library enrichment by PCR with limited cycles and sequencing using a 2x150bp Paired End configuration on the Illumina HiSeq. GL261-hCD19-luc2 and CT2A-hCD19-luc tumors were extracted from mouse brains and tumor cells were first isolated from dissociated ex vivo tumours by bead-based positive magnetic selection for hCD19. RNA was isolated using the RNeasy Mini kit. First-strand Illumina-barcoded libraries were generated using the NEB RNA Ultra Directional kit, including 12 cycles of PCR enrichment. Libraries were subsequently sequenced on an Illumina NextSeq 500 instrument using paired-end 37bp reads.