title: 3997_HN
organism: Mus musculus
description: Protocols: Mouse brains were harvested and the tumor mass was microdissected under a fluorescence-stereomicroscope (Leica). Single cells were obtained by triturating dissected GBM in PBS and subsequent treatment with 1mg/ml collagenase-I followed by several wash-steps and filtering through a 40mm cell strainer.FACS sorting was done using a MoFlo Astrios EQ,(Beckman Coulter) equipped with 7 Lasers (355nm, 405nm, 488nm, 532nm, 561nm, 592nm, 642nm) and operated with a 70mm Nozzle at 88kHz on single cell sort mode. Cells were pregated using FSC/SSC debris exclusion, FSC-W singlet discrimination and bulk sorted for living (excluding SYTOX Blue dead stained cells; ThermoFischer Scientific ) RFP+ tumor-derived vascular pericytes (n is 4 HN-treated vs. 3-aCSF control animals) or AF647-CD31+ stained (isotype-controlled) vascular endothelial cells (n is 2 animals per group). Collected cells were resuspended in 100 µL Trizol, and stored at -80 °C.   During tumor growth HN (200 nM) or aCSF was applied by intracerebral infusion via minipump.  Total RNA was extracted from cells following the Trizol Reagent User Guide (Thermo Fisher Scientific). NEBNext® Single Cell/Low Input cDNA Synthesis & Amplification Module was used for full-length cDNA synthesis and amplification, and NEBNext® Ultra™ II DNA Library Prep Kit for Illumina was used for sequencing library preparation (NEB, Ipswich, MA, USA). Briefly, cDNA was fragmented, and adaptor was added using Transposase, followed by limited-cycle PCR to enrich and add index to the cDNA fragments. The final library was assessed with Agilent 5300 Fragment Analyzer (Agilent Technologies, Palo Alto, CA, USA).