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| Status |
Public on Mar 06, 2024 |
| Title |
CnR8_IgG_merged |
| Sample type |
SRA |
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| Source name |
92-1
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| Organism |
Homo sapiens |
| Characteristics |
cell line: 92-1
cell type: Uveal Melanoma
cell cycle synchronization: unsynchronized
specifics for sample preparation: merge of two IgG samples
treatment: not applicable
spike-in: none
chip/cut&run antibody: IgG (control)
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| Treatment protocol |
The 92-1 cells were not specifically treated for the Cut&Run experiment.
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| Growth protocol |
Uveal melanoma cells (92.1) were maintained in RPMI-1640 medium supplemented with with 10% FCS and 1% penicillin/ streptomycin. Cells were cultured at 37°C at 5% CO2.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For Cut&Run sequencing, cells were washed twice and then coupled to ConA-coated magentic beads (Polysciences Europe). Cells were resuspended with antibody buffer (0.005% digitonin and 2 mM EDTA). Then cells were incubated with the antibody (see for details above) for 2 hours at RT. Cells were then washed in the presence of digitonin and 700 ng/ml MNase was added per sample for 1 hour at 4°C, followed by two more washes.
Following the initial washes, the MNase treated cells were washed once in low salt rinse buffer and then incubated in buffer containing CaCl2 and digitonin to activate the protein A-MNase. The DNA was digested for 30 min on ice and the Mnase activity was then stoped in the presence of 50 µg/ml Rnase A. DNA fragments were released at 37°C for 30 min and the decrosslinking was done for 1 hour at 50°C in the presence of Proteinase K. DNA was extracted wit phenol-chloroform and DNA was precipitated with ethanol and resuspended in TE buffer.
In some cases (Cut&Run samples where antibodies were used for H3K4me1, H3K4me3, H3K27ac and H4ac), a 1 min crosslinking with 1% formaldehyde was performed. In these cases, all buffers were used without digitonin and instead 0.05% SDS and 0.1% Triton X-100 were added to the wash buffer.
The DNA libraries were prepared with 6 ng of CUT&RUN DNA fragments, using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs) according to the manufacturer’s instructions. The libraries were PCR-amplified with 15 PCR cycles using the NEBNext Multiplex Oligos for Illumina Kit. The libraries were purified using Agencourt AMPure XP beads (Beckman Coulter). Library quality and fragment size distribution was analyzed on a Fragment Analyzer (Advanced Analytical). Sequencing was performed in single-read or paired-end mode on a NextSeq500 or NextSeq2000 platform (Illumina).
followed by sequencing.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
NextSeq 2000 |
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| Description |
CUT&RUN-seq
n/a
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| Data processing |
Base calling was performed using Illumina's CASAVA software or FASTQ generation software v1.0.0 and the quality of the generated FASTQ files was analyzed with FastQC.
For Cut&Run sequencing, fastq-files were imported to the Galaxy web-based analysis portal and reads were aligned to the human genome (hg19) using Bowtie2. All paired CUT&RUN reads were filtered by using a Phred score cutoff of 30 and mitochondrial reads were removed. Peaks were called with MACS2.
DeepTools 2 was used to create CUT&RUN profiles. First, normalized bigWig files were created using bamcoverage with a bin size of 10 and normalizing to counts per million reads mapped (CPM). BigWig files were used to compute reads centered on YAP-seq peak summits (called with MACS2) using computeMatrix. Profiles were created with plotProfile tools. CUT&RUN data were visualized with the Integrated Genome Viewer.
Assembly: hg19
Supplementary files format and content: For CUT&RUN-seq, the provided processed files are bigwig files for data display in a genome browser.
Library strategy: CUT&RUN
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| Submission date |
Mar 22, 2023 |
| Last update date |
Mar 06, 2024 |
| Contact name |
Carsten P Ade |
| Organization name |
University of Wuerzburg
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| Department |
Lehrstuhl Biochemie und Molekularbiologie
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| Lab |
AG Martin Eilers
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| Street address |
Am Hubland
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| City |
Wuerzburg |
| ZIP/Postal code |
97074 |
| Country |
Germany |
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| Platform ID |
GPL30173 |
| Series (2) |
| GSE227972 |
MY-COMP, an inhibitor of the transcriptional activity of YAP-B-MYB, identifies NEK2 as a novel mediator of YAP oncogenesis in uveal melanoma cells [CUT&RUN] |
| GSE227974 |
MY-COMP, an inhibitor of the transcriptional activity of YAP-B-MYB, identifies NEK2 as a novel mediator of YAP oncogenesis in uveal melanoma cells |
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| Relations |
| BioSample |
SAMN33864305 |
| SRA |
SRX19751396 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7111609_CnR8_IgG_merged.bigwig |
1.4 Gb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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