|
| Status |
Public on Jun 20, 2019 |
| Title |
LN229_shSOX10_ATACseq_rep2 |
| Sample type |
SRA |
| |
|
| Source name |
adherent glioblastoma-derived cell line
|
| Organism |
Homo sapiens |
| Characteristics |
subtype: RTK_I-like glioblastoma cell line
gender: male
|
| Treatment protocol |
Inducible SOX10 knockdown LN229 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. ZH487 cells constitutively expressing SOX10-targeting shRNA were established by trasnfection with the pLKO1 plasmid bearing the SOX10 shRNA.
|
| 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 |
genomic DNA |
| Extraction protocol |
ATAC-seq was performed in biological duplicates, as described in http://msb.embopress.org/content/15/5/e8339 Briefly: viable frozen cells were incubated with Tn5 in 0.1% Igepal CA-630 (37C, 30'). Transposition was stopped with EDTA and DNA purifired using AMPure beads.
After DNA purification, barcodes were added using PCR and DNA re-purified again on AMPure beads. These prepared libraries were then sequenced (50 bp, single-end) on an Illumina HiSeq 2000 or 4000.
|
| |
|
| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
ATAC-seq datasets were processed using a custom pipeline implemented in Snakemake. Briefly, reads were trimmed using the Trimgalore tool (https://github.com/FelixKrueger/TrimGalore) and aligned using Bowtie with standard parameters. Duplicates and multi-mapping reads were removed using the samtools package and the XS flag in the bam files.Peaks were called using the callpeak mode in MACS2 (https://github.com/taoliu/MACS) for broad and narrow peaks.Various QC parameters (FRiP, PCR bottleneck coefficient, cross-strand correlation) were determined according to the ENCODE guidelines.
A consensus peakset was defined from the two biological replicates by taking the merged peaks generated by the findOverlapsOfPeaks function from the R/Bioconductor package ChIPpeakAnno. ATAC signal was calculated for these peaks using bigWigAverageOverBed.
Differentially "bound" peaks were identified using the R/Bioconductor package DiffBind.
Genome_build: hg19
Supplementary_files_format_and_content: bigWig genome-wide coverage tracks; MACS narrowPeak calls
|
| |
|
| Submission date |
Jun 19, 2019 |
| Last update date |
Jun 20, 2019 |
| 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) |
| GSE121723 |
Glioblastoma epigenome profiling identifies SOX10 as a master regulator of molecular tumour subtype |
| GSE133040 |
Glioblastoma epigenome profiling identifies SOX10 as a master regulator of molecular tumour subtype - cell line ATACseq |
|
| Relations |
| BioSample |
SAMN12097130 |
