2024-07-15
ENA first public
ERC000011
ENA-CHECKLIST
Neurosurgical Research Department of Neurosurgery University Hospital, LMU Munich
INSDC center name
public
INSDC status
ERS19606498
SRA accession
E-MTAB-14064:3929_aCSF
Submitter Id
90
day
age
ArrayExpress
broker name
pericyte
cell_type
not collected
collection date
house mouse
common name
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).
description
adult
developmental stage
wild type genotype
genotype
not collected
geographic location (country and/or sea)
3929
individual
not applicable
isolate
Mus musculus
organism
Mus musculus
organism
brain tumor
organism part
Mus musculus
scientific_name
male
sex
PDGFRb-cre::ER2, R26-RFP
strain
3929_aCSF
title