|
| Status |
Public on Jan 29, 2018 |
| Title |
SHEP21_2HR_H3K4ME3 |
| Sample type |
SRA |
| |
|
| Source name |
Neuroblastoma
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: SHEP-21N
input: SHEP21_24HR_INPUT
treatment: 0.2 ug/mL DOX for 24HR
growth protocol: 10% Tet-Free FBS, RPMI
barcode: CAGATCTG
antibody: H3K4me3
antibody maker: EMD Millipore
antibody catalog number: 07-473
|
| Treatment protocol |
Tet-Off MYCN shutdown was performed by addition of doxycycline (0.2μg/mL) to growth media
|
| Growth protocol |
SHEP-21N cells were kindly provided by Dr. William Weiss (University of California, San Francisco) and cultured in RPMI supplemented with 10% Tetracycline-Free FBS (Clontech). Tet-Off MYCN shutdown was performed by addition of doxycycline (0.2μg/mL) to growth media
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP-Rx was performed as described previously substituting mouse embryonic stem cells in place of Drosophila S2 cells (Orlando et al., 2014). Mouse embryonic stem (ES) cells were grown to 75% confluence and cross-linked with 1% formaldehyde followed by quenching (125 mM glycine) as described in the previous section. Cells were washed in cold PBS and harvested by cell scraper in cold PBS with protease inhibitors (Roche). Cells were centrifuged at 1650 x g for 5 minutes and flash frozen and stored at -80°C 10E06 cells per pellet. Fixed mouse ES cell pellets were resuspended in a cytosolic lysis buffer and then spiked directly into the cytosolic lysate of fixed neuroblastoma cells at a ratio of 5% of the total number of cells. The lysate (neuroblastoma cells spiked with mouse ES cells) was then carried through the ChIP protocol.
Libraries were prepared using the Rubicon Thruplex FD library preparation kit (cat#: 400427) per manufacturers instructions.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
Spike in normalized ChIP-Seq for H3K4me3 in SHEP21-N, 2hr after doxycycline treatment
|
| Data processing |
Sequenced reads were aligned to a custom reference bowtie2 index combinging the HG19 and MM9 genome builds
ChIP_RX scaling factors were determined as in Orlando et al., 2014, "Quantitative ChIP-Seq normalization reveals global modulation of the epigenome", PMID 25437568
Peaks were called using MACS1.4.2 with p-val =1e-9 and background datasets as described in characteristic: input
Genome_build: hg19:mm9
Supplementary_files_format_and_content: Processed ChIP-Rx datasets are provided in a cell count normalized scaled bedgraph format. File HG19_SHEP21_SCALE.txt contains ChIP-Rx scale factors for each dataset
|
| |
|
| Submission date |
Apr 10, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
James Bradner |
| E-mail(s) |
bradner_computation@dfci.harvard.edu
|
| Organization name |
Dana-Farber Cancer Institute
|
| Department |
Medical Oncology
|
| Lab |
Bradner Lab
|
| Street address |
450 Brookline
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02215 |
| Country |
USA |
| |
|
| Platform ID |
GPL18573 |
| Series (2) |
| GSE80150 |
Enhancer invasion shapes MYCN dependent transcriptional amplification in neuroblastoma [ChIP_RX] |
| GSE80154 |
Enhancer invasion shapes MYCN dependent transcriptional amplification in neuroblastoma |
|
| Relations |
| BioSample |
SAMN06704923 |
| SRA |
SRX2731779 |
