|
| Status |
Public on Jan 27, 2021 |
| Title |
mGB1 WES |
| Sample type |
SRA |
| |
|
| Source name |
mGB line, after first in vivo passage
|
| Organism |
Mus musculus |
| Characteristics |
tissue: mouse glioblastoma cell line
|
| Treatment protocol |
tNSCs were isolated from the subventricular zone (SVZ) of mice 2 weeks after the injection of either tamoxifen or oil. For mGB tumor cell isolation, glioma-bearing mice were euthanized with carbon dioxide; brains were minced and dissociated in Leibovitz-L15 (Life Technologies) containing 10 U/mL papain, 5 mM EDTA, and 200 U/mL DNAse. Cells were grown according in stem cell-maintaining conditions.
|
| Growth protocol |
Tlx-CreERT2/p53-floxed/pten-floxed (double knockout [DKO]) mice were described in Costa et al., Blood Advances 2019. To induce recombination of floxed alleles, 4-week-old mice were injected intraperitoneally with 1 mg tamoxifen (S5007 Sigma) in 5% ethanol and 95% oil (T5648 Sigma) for 5 consecutive days. Animal experiments were approved by the German responsible authority (Regierungspräsidium Karlsruhe) and performed in conformity with the German law for Animal Protection (animal license number: G-156/15, G-199/11). Control NSCs were isolated from the subventricular zone.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA and RNA were isolated using spin column preparation kits from cells cultured in vitro.
Genomic DNA was fragmented to a size range of 150-250 bp using the Covaris S220 instrument. The fragmented DNA was end repaired and adenylated. The SureSelect Adaptor was then ligated followed by a pre-amplification. After library preparation target regions were captured by hybridization of biotinylated baits (library probes from the Agilent SureSelect XT Mouse All Exon). Captured target sequences were then isolated using streptavidin coated magnetic beads. Subsequently the appropriate 8 bp single index tags were added during sequencing library amplification. All steps were done using Agilent SureSelect XT Reagent Kit. Final quality control was done using Qubit 3 fluorometer and Agilent Bioanalyzer 2100. The libraries were sequenced in paired-end mode (2 x 50 nt) on a NovaSeq6000 S2 Flowcell resulting in ~200 million distinct sequencing reads per library.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
mouse glioblastoma cells from gross tumour generated in C57/Bl6N genetically engineered mouse model using double knock-out of Pten and Trp53, first in vivo passage
|
| Data processing |
Whole exome sequencing paired end reads in fastq format were aligned using BWA (v0.7.15) to the mm10 reference genome with options: -M –T 0. Duplicates were marked using sambamba (0.6.6).
CNV analysis was performed using CNVkit (v0.9.6). Tumor WES samples (tNSC0-3, mGB0-2) were analysed with reference to the two normal WES samples, ctrlNSC and normal splenocytes. The mm10 genome .fasta was used to calculate accessibility as follows: access mm10.fa -s 10000 –o mm10_accessibility.bed. The S0276129 bedfile with Agilent capture array loci, lifted over to the mm10 genome, was used as the target regions. Regions were annotated using UCSC’s mm10 flat reference (http://hgdownload.soe.ucsc.edu/goldenPath/mm10/database/refFlat.txt.gz, accessed 21 November 2019).
Calling was performed with the batch command and the following options: batch TUMOR_SAMPLES --normal NORMAL_SAMPLES --targets S0276129_mm10.bed --fasta mm10.fa --access mm10_accessibility.bed --annotate refFlat.txt --drop-low-coverage. Copy number state segmentation was performed using the ‘cbs’ method as implemented in the R package DNAcopy (v1.54.0).
The results of the CNV calling were visualised using the R package circlize (v0.4.4)35in R v3.5.1. Regions overlapping annotated segmental duplications (downloaded from the UCSC Table Browser, accessed 25 January 2020) were removed before plotting due to potential issues with inferring copy number states in these repetitive regions. For ease of visualisation, extreme values with CNVkit log2 copy ratio < -2 or > 2 were set to this minimum and maximum. The integer CNVkit segmented copy number states were smoothed using the rollarray command from zoo (v1.8-2) with the options width = 301, partial=TRUE.
Genome_build: mm10
Supplementary_files_format_and_content: GB_GEMMs_mGB_tNSC_CNVkit_results.txt.gz: table of output of CNV calling results, generated by CNVkit
|
| |
|
| Submission date |
Feb 19, 2020 |
| Last update date |
Jan 27, 2021 |
| Contact name |
Bernhard Radlwimmer |
| E-mail(s) |
b.radlwimmer@dkfz-heidelberg.de
|
| Organization name |
Deutsches Krebsforschungszentrum / German National Cancer Research Centre
|
| Department |
Department of Molecular Genetics
|
| Street address |
Im Neuenheimer Feld 280
|
| City |
Heidelberg |
| ZIP/Postal code |
69120 |
| Country |
Germany |
| |
|
| Platform ID |
GPL24247 |
| Series (2) |
| GSE145558 |
A novel neural stem cell-derived immunocompetent mouse model of glioblastoma for preclinical studies (WES) |
| GSE145559 |
A novel neural stem cell-derived immunocompetent mouse model of glioblastoma for preclinical studies |
|
| Relations |
| BioSample |
SAMN14137868 |
| SRA |
SRX7750558 |
