|
| Status |
Public on Jun 08, 2016 |
| Title |
Input on DMSO-treated cells |
| Sample type |
SRA |
| |
|
| Source name |
A375 melanoma cell line
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: A375
antibody: none
|
| Treatment protocol |
One hundred million A375 cells per condition were treated with DMSO or 25 μM A771726. After a 48 hr treatment, cells were fixed in 11% formaldehyde and subjected to chromatin immunoprecipitation with HEXIM1 antibody (Abcam) or Cdk9 (C-20) antibody (Santa Cruz).
|
| Growth protocol |
Human A375 malignant melanoma cells (ATCC) are grown on standard tissue culture plates in filter sterilized DMEM (Life Technologies) with 10% heat-inactivated FBS (Atlanta Biologicals), 1X GlutaMAX (Life Technologies) and 1% Penicillin-Streptomycin (Life Technologies).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
After chromatin immunoprecipitation, cross-link reversal is carried out and precipitated DNA is purified by phenol:chloform:isoamyl alcohol extraction.
The End-It DNA End-Repair Kit (Epicentre) was used to turn DNA overhangs into phosphorylated blunt ends and the Agencourt AMPure XP PCR Purification Kit (Beckman Coulter) was used to purify the resulting samples using a 1.8X ratio of beads to sample. Next, a single A in the 3’ end was added to samples using the Klenow Fragment enzyme (NEB) to allow for directional ligation and the AMPure Kit was again used to purify samples. Illumina adaptor oligos (1:10 dilution) were added to samples using the Quick Ligation Kit (NEB) and NEBNext Multiplex Oligos for Illumina Kit (NEB), and samples were purified with the AMPure Kit. Samples were then size-selected with the AMPure beads with a 0.9X bead to sample ratio. Multiplexing primers from the NEBNext Multiplex Oligos Kit were added during the 18-cycle PCR enrichment step, which utilized the Phusion High-Fidelity PCR Master Mix (NEB), to generate indexed libraries. Indexed libraries were separated on a 2% agarose gel. Products between 150-350 base pairs were excised and gel purified using the QIAquick Gel Extraction Kit (Qiagen). The concentration of the isolated libraries was estimated with a high-sensitivity DNA chip from Agilent according to manufacturer’s protocol and libraries were mixed in equal quantities and sequenced on Illumina Hi-Seq2000. Index sequences from multiplexed primers were used to identify treatment group and reads were demultiplexed using a perl script.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
ChIP-Seq aligned to hg19 with Bowtie with max mismatch 2bp
peaks were called using MACS 2.0 with the following setting: q-value 0.05 bigwig file converted from bedGraph pileup from MACS 2.0 output
Genome_build: hg19
Supplementary_files_format_and_content: bigwig file converted from bedGraph pileup from MACS 2.0 output
|
| |
|
| Submission date |
Sep 28, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Leonard Zon |
| E-mail(s) |
zon@enders.tch.harvard.edu
|
| Organization name |
Boston Children's Hospital
|
| Department |
Oncology/Hematology
|
| Street address |
1 Blackfan Circle
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
| |
|
| Platform ID |
GPL16791 |
| Series (2) |
| GSE61825 |
Emergence of neural crest identity is a key early step in melanoma formation [ChIP-Seq] |
| GSE75356 |
A zebrafish melanoma model reveals emergence of neural crest identity during melanoma initiation |
|
| Relations |
| BioSample |
SAMN03083467 |
| SRA |
SRX712803 |
