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GEO help: Mouse over screen elements for information. |
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| Status |
Public on May 13, 2019 |
| Title |
BT245-KO-C2 |
| Sample type |
SRA |
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| Source name |
Tumor-derived cell-line
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| Organism |
Homo sapiens |
| Characteristics |
genotype: H3.3K27M-KO
treatment: None
tissue: glioma tumor
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from cell pellets using the RNeasy mini kit (Qiagen) according to instructions from the manufacturer.
Library preparation was performed with ribosomal RNA (rRNA) depletion according to instructions from the manufacturer (Epicentre) to achieve greater coverage of mRNA and other long non-coding transcripts. Paired-end sequencing was performed on the Illumina HiSeq 2000, 2500 and 4000 platforms.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
Data processing. Adaptor sequences and the first four nucleotides of each read were removed from the read sets using Trimmomatic (v0.32). Reads were scanned from start to end and truncated if and when the average quality of a 4-nucleotide sliding window fell too low (phred33<30). Short reads (<30 bp) were subsequently discarded. Multiple control metrics were obtained using FASTQC (v0.11.2), samtools (v0.1.19), BEDtools (v2.17.0) and custom scripts.
Gene expression analysis. The remaining clean set of reads were then aligned to the reference genome build hg19 (GRCh37) with STAR (v2.3.0e) using the default parameters. Multimapping reads (MAPQ<1) were discarded from downstream analyses. Gene expression levels were estimated by quantifying uniquely mapped reads falling into exonic regions defined by the ensGene annotation set from Ensembl (GRCh37; N=60234 genes) using featureCounts (v1.4.4). Normalization (mean-of-ratios), variance-stabilized transformations of the data, as well as differential expression analysis, were performed using DESeq2. Unless otherwise stated, all reported p-values have been adjusted for multiple testing using the Benjamini-Hochberg procedure.
Repeat element expression analysis. The clean set of rRNA-depleted RNA-seq reads were aligned to the human repeat genome, as previously described. The reference repeat sequences were downloaded from Repbase (v23.03) (http://www.girinst.org/repbase). We then combined humrep.ref and humsub.ref into a single reference of repeat sequences for the human genome, covering a total of 1132 consensus repeat element sequences. All subsequent steps closely mirror those detailed for gene expression analysis. Normalization (library size) factors derived from canonical genes (Ensembl ensGene annotation) using mean-of-ratios as described above were used to normalize repeat elements.
Genome_build: hg19
Supplementary_files_format_and_content: Raw abundance measurements of Ensembl genes
Supplementary_files_format_and_content: Raw abundance measurements of Repbase repeat elements
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| Submission date |
Jul 20, 2018 |
| Last update date |
May 13, 2019 |
| Contact name |
Nada Jabado |
| Organization name |
McGill University
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| Department |
Department of Pediatrics
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| Lab |
Jabado Lab
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| Street address |
1001 Décarie Boulevard
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| City |
Montreal |
| State/province |
Québec |
| ZIP/Postal code |
H4A 3J1 |
| Country |
Canada |
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| Platform ID |
GPL20301 |
| Series (2) |
| GSE117446 |
Pervasive H3K27 acetylation leads to ERV expression and a therapeutic vulnerability in H3K27M gliomas [RNA-Seq] |
| GSE128745 |
Pervasive H3K27 acetylation leads to ERV expression and a therapeutic vulnerability in H3K27M gliomas |
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| Relations |
| BioSample |
SAMN09697983 |
| SRA |
SRX4413872 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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