|
| Status |
Public on Jan 27, 2021 |
| Title |
mGB0 RNA-Seq |
| Sample type |
SRA |
| |
|
| Source name |
mGB line, initial isolation
|
| Organism |
Mus musculus |
| Characteristics |
tissue: mouse glioblastoma cell line
|
| Treatment protocol |
NSCs were isolated from the subventricular zone (SVZ) of mice 2 weeks after the injection of either tamoxifen or oil. For 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).
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
DNA and RNA were isolated using Qiagen spin column preparation kits from cells cultured in vitro.
For RNA-seq: poly(A)-positive mRNA transcripts were isolated from the total RNA by binding to magnetic oligo(d)T beads (RNA purification beads). After elution from the beads and fragmentation, the mRNA was reverse transcribed during first and second strand synthesis yielding double-stranded cDNA. The ends of the cDNA molecules were end repaired and an adenosine overhang was added to all 3’ ends (A-tailing step) to facilitate the ligation of the adapter molecules. These adapters have individual dedicated index sequences and add the Illumina specific sequences that are needed for amplification, flow cell hybridization and sequencing. 8 bp single-index NEXTflex DNA barcodes (Bioo Scientific) were used. The final ligation product was amplified via PCR. Double-stranded cDNA sequencing libraries were further checked for quality and quantity 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 ~50 million distinct sequencing reads per library.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
mouse glioblastoma cells isolated from gross tumour generated in C57/Bl6N genetically engineered mouse model using double knock-out of Pten and Trp53, initial isolation
GB_GEMMs_mGB_tNSC_log-cpm_expression_Gencode_M2.csv.gz
GB_GEMMs_Idh1-Idh2_hotspots_SNV_calling_RNAseq.vcf
|
| Data processing |
Reads were aligned to the mouse genome (mm10 build) with the Gencode reference transcriptome (M2) using STAR (v2.5.2a) with the following options: --outFilterMismatchNmax 2 --outFilterMismatchNoverLmax 0.05 --alignIntronMax 1 --outFilterMatchNminOverLread 0.95 --outFilterScoreMinOverLread 0.95 --outFilterMultimapNmax 10 --outFilterIntronMotifs RemoveNoncanonical --outFilterType BySJout --outSAMunmapped Within --outSAMattributes Standard --alignIntronMin 21 --outFilterMatchNmin 16.
Overlapping read pairs were clipped using the clipOverlap tool in bamUtil (v1.0.9) 37. The raw counts matrix was generated using featureCounts (v1.5.3)38 using options -Q 255 -p -t exon against the M2 transcriptome. Aligned data were quality controlled using RSeQC (v2.6.4)39.
Gene expression values for each sample were quantified using limma-voom (v3.34.4)'s log-cpm metric.
Single nucleotide variants (SNVs) were called using BCFTools (v1.9) at the 3 hotspots (Idh1: R132 = 1:65170977-65170979; Idh2: R140 = 7:80099112-80099114; R172 = 7:80099016-80099018, mm10 coordinates). bcftools mpileup was used on the full set of samples for each data type, and consensus calling performed with bcftools call –c.
Genome_build: mm10
Supplementary_files_format_and_content: GB_GEMMs_mGB_tNSC_log-cpm_expression_Gencode_M2.csv.gz: limma-voom calculated log-cpm values of gene expression
Supplementary_files_format_and_content: GB_GEMMs_Idh1-Idh2_hotspots_SNV_calling_RNAseq.vcf - SNV calling results for Idh1/2 mutation hotspots
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| |
|
| 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) |
| GSE145557 |
A novel neural stem cell-derived immunocompetent mouse model of glioblastoma for preclinical studies (RNA-Seq) |
| GSE145559 |
A novel neural stem cell-derived immunocompetent mouse model of glioblastoma for preclinical studies |
|
| Relations |
| BioSample |
SAMN14137877 |
| SRA |
SRX7750549 |
