|
| Status |
Public on Nov 05, 2018 |
| Title |
LN229_shControl_RNAseq |
| Sample type |
SRA |
| |
|
| Source name |
adherent glioblastoma-derived cell line
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: LN229
cell type: adherent glioblastoma-derived cell line
subtype: RTK_I cell-like line
gender source: male
genotype/variation: pLKO-Tet-On non-targeting (nt) shRN
|
| Treatment protocol |
Inducible SOX10 knockdown cells were established by infecting cells with pLKO-Tet-On non-targeting (nt) shRNA and pLKO-Tet-On SOX10 shRNA (TRCN0000018988) lentiviral particles and puromycin selection (1 µg/ml) for 7 days. shRNA expression was induced by adding 0.1 µg/ml doxycycline to the medium for at least 7 days.
|
| Growth protocol |
The human glioblastoma cell line LN229 was obtained from ATCC (cat. CRL-2611). Cells were cultured in DMEM containing 1 g/L glucose (D5921, Sigma) supplemented with 10% tetracycline-free fetal bovine serum (Clontech), 1% penicillin and streptomycin (P/S) mix, and glutamine (0.5 mM). The ZH487 glioblastoma spheroid primary cells were originally established at the University of Zurich Hospital. ZH487 cells were cultured in Neurobasal medium (Cat. 12348017, NBM) supplemented with 2% B27 (cat. 12587010, retinoic acid-free, Invitrogen), EGF (20 ng/ml, AF-100-15, Peprotech), FGF (20 ng/ml, cat. 100-18B, Peprotech) and glutamine (0.5 mM). DNA methylation array (Illumina EPIC) analysis classified the ZH487 primary tumour and derived cell line as part of the RTK I subtype. All cell lines were cultured under 10% CO2 at 37℃ with humidity. Cell identities were verified by the Multiplex human Cell line Authentication Test (MCA). Cells were also tested for mycoplasma contamination with the Multiplex cell test (Multiplexion GmbH, Friedrichshafen, Germany).
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA was isolated using the RNeasy Microkit (Qiagen) according to the manufacturer's standard protocol.
Libraries were constructed using the Illumina TruSeq RNA Sample Preparation Kit v2 according to the manufacturer's instructions. Briefly, 1000ng of total RNA was used for the polyA-based selection and fragmented. The first-strand cDNA was synthesized with random hexamer and SuperScript II (Invitrogen) following with second-strand synthesis, then the double-stranded cDNA fragments were end-repaired, A-tailed on the 3′ end, ligated to indexed adapters and amplified with 6 cycles of PCR. The final libraries were validated using Qubit (Invitrogen) and Agilent 2100 Bioanalyzer (Agilent Technologies). Subsequently, the libraries were diluted to 10nM and pooled in equimolar ratios. The pooled library was sequenced with the Illumina HiSeq2000 (50 bp single-end) system.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
Reads were aligned to the human genome (hg19 build) with the Gencode reference transcriptome (v19) using STAR (v2.3.0e).
Read counts for each gene were quantified as the total number of reads mapping to exons using htseq-count (0.6.0).
bigWig coverage tracks were produced using deepTools2.
Genome_build: hg19
Supplementary_files_format_and_content: bigWig coverage tracks
|
| |
|
| Submission date |
Oct 24, 2018 |
| Last update date |
Nov 05, 2018 |
| 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 |
GPL11154 |
| Series (2) |
| GSE121717 |
Glioblastoma epigenome profiling identifies SOX10 as a master regulator of molecular tumour subtype - cell line RNAseq experiments |
| GSE121723 |
Glioblastoma epigenome profiling identifies SOX10 as a master regulator of molecular tumour subtype |
|
| Relations |
| BioSample |
SAMN10285472 |
