|
| Status |
Public on Apr 08, 2016 |
| Title |
HEXIM1 ChIP on A771726-treated cells |
| Sample type |
SRA |
| |
|
| Source name |
A375 melanoma cell line
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: A375
cell type: malignant melanoma
chip antibody: HEXIM1 (Abcam)
|
| Treatment protocol |
One hundred million A375 cells per condition were treated with DMSO, 25 μM leflunomide or 25 μM A771726. Drugs used were leflunomide (Enzo Life Sciences) and A771726 (Enzo Life Sciences). After a 48 hr treatment, cells were washed, proteins and DNA were cross-linked with 11% formaldehyde and cell pellets were frozen down at -80C.
|
| 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 |
DNA was sonicated and chromatin immunoprecipitation was performed with either HEXIM1 (Abcam) or CDK9 (Santa Cruz) antibodies. DNA was then de-cross-linked from pulled down protein and purified by phenol:chloroform:isoamyl alcohol extraction.
The NEBNext multiplex oligos for Illumina Kit (NEB) was used to make the samples suitable for multiplexing. The PCR reaction was run for 18 cycles. The indexed libraries were purified on a 2% agarose gel, and were cut out between 150 and 350 bp. 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
Genome_build: hg19
Supplementary_files_format_and_content: bedGraph pileup from MACS 2.0 output
|
| |
|
| Submission date |
May 08, 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) |
| GSE57431 |
HEXIM1 is induced by DHODH inhibition to suppress melanoma [ChIP-Seq] |
| GSE57432 |
HEXIM1 is induced by DHODH inhibition to suppress melanoma |
|
| Relations |
| Alternative to |
GSM1661789 (duplicate) |
| BioSample |
SAMN02767794 |
| SRA |
SRX535456 |
