|
| Status |
Public on Jun 20, 2019 |
| Title |
LN229_shControl_BRD4 |
| Sample type |
SRA |
| |
|
| Source name |
adherent glioblastoma-derived cell line
|
| Organism |
Homo sapiens |
| Characteristics |
subtype: RTK_I cell-like line
gender: male
chip antibody: BRD4
chip antibody vendor: Bethyl Laboratories
chip antibody cat.#: A301-985A100
|
| 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 |
Cells were cross-linked with 1% methanol-free formaldehyde for 15 min and quenched with 0.125M glycine. Chromatin was isolated by adding lysis buffer and Dounce homogenization. Collected chromatin was sheared via sonication to an average length of 300-500bp. Input genomic DNA was prepared from collected chromatin by treatment with RNase, proteinase K and de-crosslinking under heat, and then isolated by ethanol precipitation. Pellets were resuspended and DNA quantified on a NanoDrop spectrophotometer. Estimated total chromatin yield was calculated based on this amount. 30ug of chromatin was pre-cleared with protein A agrose beads (Invitrogen), and DNA precipitated using 4ug of antibody against SOX10 or BRD4. This DNA was isolated from the beads by washing followed by SDS buffer elution, RNase/proteinase K treatment and de-crosslinking under heat (65C overnight incubation). DNA was then purified using phenol-chloroform extraction and ethanol precipitation.
Sequencing libraries were prepared from ChIPped and input DNA via standard protocols (enzymatic end-polishing, dA-addition and adaptor ligation) on an Apollo 342 automated system (Wafergen Biosystems/Takara).
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
ChIP-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. Input control and corresponding IP datasets were scaled using the SES method and converted into a bigwig track using the bamCompare tool of the deepTools2 suite. Peaks were called using the callpeak mode in MACS2 (https://github.com/taoliu/MACS) for broad and narrow peaks. In addition, SICER was used to call peaks using the gap 600 and window 200 parameters. Various QC parameters (FRiP, PCR bottleneck coefficient, cross-strand correlation) were determined according to the ENCODE guidelines. In addition, visual QC was performed using the signal profile at TSS of annotated genes and the fingerprint method from the DeepTools2 suite.
Genome_build: hg19
Supplementary_files_format_and_content: bigWig tracks of SES-normalised coverage; bed files of MACS2 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) |
| GSE121716 |
Glioblastoma epigenome profiling identifies SOX10 as a master regulator of molecular tumour subtype - cell line ChIPseq experiments |
| GSE121723 |
Glioblastoma epigenome profiling identifies SOX10 as a master regulator of molecular tumour subtype |
|
| Relations |
| BioSample |
SAMN12097124 |
