|Public on Jan 01, 2017
|HEXIM1>CDK9 reChIP on A375 cells
|A375 melanoma cell line
|cell line: A375
|One hundred million A375 cells per condition were washed, proteins and DNA were cross-linked with 11% formaldehyde and cell pellets were frozen down at -80C.
|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).
|DNA was sonicated and chromatin immunoprecipitation was performed with either HEXIM1 (Abcam) followed by CDK9 (Santa Cruz) antibodies sequentially and vice versa. 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.
|Illumina HiSeq 2500
|DMSO HEXIM1>CDK9 reChIP
|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
Supplementary_files_format_and_content: bedGraph pileup from MACS 2.0 output
|Apr 20, 2015
|Last update date
|May 15, 2019
|Boston Children's Hospital
|1 Blackfan Circle
|Nucleotide stress induction of HEXIM1 suppresses melanoma by modulating cancer cell-specific gene transcription [reChIP-seq]
|Nucleotide stress induction of HEXIM1 suppresses melanoma by modulating cancer cell-specific gene transcription