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Sample GSM1382442 Query DataSets for GSM1382442
Status Public on Apr 08, 2016
Title CDK9 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: CDK9 (Santa Cruz)
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 GSM1661787 (duplicate)
BioSample SAMN02767792
SRA SRX535454

Supplementary file Size Download File type/resource
GSM1382442_CDK9_A771.MACS2_in_Galaxy_treat_pileup.bedGraph.gz 129.0 Mb (ftp)(http) BEDGRAPH
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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