|
| Status |
Public on Apr 21, 2023 |
| Title |
CDK13_W878L_PolII |
| Sample type |
SRA |
| |
|
| Source name |
Melanoma
|
| Organism |
Danio rerio |
| Characteristics |
tissue: Melanoma
chip antibody: RNA Pol II
antibody manu.: Abcam
antibody catalog: ab817 (8WG16)
antibody lot: lot GR313984-17
cdk13 status: W878L
sg target: N/A
|
| Growth protocol |
p53-/-; mitfa:BRAFV600E;Na-/- one-cell embryos were injected with either 20ng/uL control or experimental MiniCoopR (MCR) DNA along with tol2 in vitro transcribed RNA for integration. In all experiments, 20 zebrafish were raised per tank to control for density effects. Zebrafish were scored for the emergence of raised melanoma lesions as published. Zebrafish were sacrificed on ice and the tumors were dissected. 11 EGFP or 6 CDK13 W878L expressing melanomas were isolated between 18-19 weeks post fertilization. Tumors were combined and minced. Cells were collected and fixed in 1.1% formaldehyde solution for 10min at room temperature (RT). Tumors were homogenized and filtered through a 100um filter, spun down, washed with 1X PBS, and cells were counted using a Neubauer Chamber (9.00E+07 for CDK13 W878L and 7.10E+07 for EGFP expressing melanomas). For anti-CDK13 ChIP-sequencing from melanomas from mitfa:BRAFmut+/+; p53-/-; Na-/- zebrafish expressing either CDK13 R860Q + ccnT1 CRISPR or CDK13 R860Q + ctrl CRISPR as above, 4 melanomas were isolated from each condition at the same time point (19.1 weeks post fertilization) and the above protocol was replicated (Neubauer Chamber 7.90 E-7 for R860Q + ccnT1 CRISPR and 8.5 E-7 for R860Q + ctrl CRISPR.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Libraries were performed using the NEBNextMultiplex Oligos for Illumina kit (NEB) according to instructions. Samples were sequenced with 100bp single end reads.
Nuclear cell extracts were sonicated to solubilize the chromatin. The chromatin extracts containing DNA fragments with an average size of 200-400 bp were immunoprecipitated using different antibodies.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
Raw reads were aligned twice: first to the dm6 revision of the D. melanogaster genome to remove exogenous spike-in reads using bowtie version 1.2.2 with parameters -k 1 -m 1; unmapped reads were then aligned to the danRer10 revision of the zebrafish genome.
danRer10
Supplementary_files_format_and_content: bigWig files were created from the alignment bam file using the R with the GenomicAlignments package and with gr.cov and export.bw from rtracklayer
|
| |
|
| Submission date |
May 16, 2019 |
| Last update date |
Apr 21, 2023 |
| Contact name |
Richard A Young |
| E-mail(s) |
young_computation@wi.mit.edu
|
| Phone |
617-258-5219
|
| Organization name |
Whitehead Institute for Biomedical Research
|
| Lab |
Young Lab
|
| Street address |
9 Cambridge Center
|
| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02142 |
| Country |
USA |
| |
|
| Platform ID |
GPL14875 |
| Series (2) |
| GSE131332 |
Oncogenic CDK13 Mutations Impede Nuclear RNA Surveillance (zfChIPseq) |
| GSE131334 |
Oncogenic CDK13 Mutations Impede Nuclear RNA Surveillance |
|
| Relations |
| BioSample |
SAMN11659633 |
| SRA |
SRX5846407 |
