 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Nov 05, 2018 |
| Title |
LN229_shControl_H3K36me3 |
| Sample type |
SRA |
| |
|
| Source name |
LN229_shControl
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: LN229
cell type: adherent glioblastoma-derived cell line
subtype: RTK_I cell-like line
gender source: male
chip antibody: H3K36me3
chip antibody vendor: Abcam
chip antibody cat.#: ab9050
molecule subtype: genomic DNA and associated chromatin
|
| 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 |
genomic DNA |
| Extraction protocol |
Cells were cross-linked with 1% methanol-free formaldehyde for 10 min. After quenching with glycine, cells were washed three times with PBS and the cell pellet was treated with 4U MNase per 1x106 cells for 15 min. MNase was stopped with 10x Covaris buffer and the chromatin was sheared for an additional 15 min with the LE220 Covaris device. The soluble chromatin was then recovered, quantified and 2µg chromatin was used in each IP with 2µl of antibody (H3: ab1791; H3K4me1, ab8895; H3K4me3, ab8580; H3K9me3, ab8898; H3K27ac, ab4729; H3K36me3, ab9050; H3K27me3, ab6002; all Abcam). After IP and washes with Covaris buffer, Li-buffer and TE, precipitated chromatin was digested with proteinase K and purified with AMPure beads.
Illumina sequencing libraries were prepared from purified DNA with the NEBNext Ultra library preparation kit (NEB) according to standard protocols.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
processed data file:
LN229_shControl_ChromHMM_18states.bed
LN229_shControl_ChromHMM_simplified_7states.bed
|
| 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 (WGS) 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.
Chromatin segmentation was defined using the ChromHMM tool. ChIP-seq data (H3K27ac, H3K27me3, H3K36me3, H3K4me1, H3K4me3, H3K9me3) and WGS data (input control) were binarized using ChromHMM’s “BinarizeBam” command. The Epigenome Roadmap 18-state model was used to segment the genome of each sample. The consensus state for a subtype is defined as the state with the highest frequency in a given segment, and a minimum frequency of 50%.
The simplified 7-state model was defined by mapping the 18 states as follows: 1-4 to TSS (Tss), 5-6 to Transcription (Tx), 7-11 and 15 to Enhancer (Enh), 12-13 to Heterochromatin/Repeats (Het/Rpts), 14 to Bivalent TSS (TSSBiv), 16-17 to Polycomb Repressed (ReprPC), 18 to Quiescent (Qui).
genome build: hg19
processed data files format and content: ChromHMM results for each sample are contained in two bedfiles: the full 18-state and the reduced 7-state model. For each IPed factor, 3 files are included: a bigWig coverage file and two peak call bedfiles.
|
| |
|
| Submission date |
Oct 24, 2018 |
| Last update date |
Nov 15, 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) |
| 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 |
SAMN10285446 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3444410_LN229_shControl_H3K36me3_W200-G600-FDR0.01-island.bed.gz |
262.9 Kb |
(ftp)(http) |
BED |
| GSM3444410_LN229_shControl_H3K36me3_coverage_SES_subtract.bigWig |
230.0 Mb |
(ftp)(http) |
BIGWIG |
| GSM3444410_LN229_shControl_H3K36me3_peaks.narrowPeak.bed.gz |
1.6 Mb |
(ftp)(http) |
BED |
| Raw data not provided for this record |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
|
|
|
|
 |
