GSM5034047: MCF-7 ChIP-seq HDAC6 PRL+ ACY241- biological rep 2 technical rep 1; Homo sapiens; ChIP-Seq
ChIP-seq:  Approximately 1.0 x 108 cells were used for each experimental condition and biological replicate (n=2 per condition and ChIP target). At the conclusion of ACY-241 treatment and/or prolactin stimulation, chromatin was cross-linked in 1% formaldehyde for 12 minutes, followed by neutralization with 1/20 volume of 2.5 M glycine. Cells were washed twice with ice-cold PBS, scraped and collected in a third volume of PBS, and briefly spun down before pellets were snap-frozen in liquid nitrogen. With the exception of sonication, which was performed using the Covaris M220 Focused-ultrasonicator (Covaris, Inc.), chromatin isolation, immunoprecipitation, and DNA extraction were performed as previously described [Lee, T.I., S.E. Johnstone, and R.A. Young, Chromatin immunoprecipitation and microarray-based analysis of protein location. Nat Protoc, 2006. 1(2): p. 729-48]. DNA purity was assessed by spectrophotometry at 260, 270, and 280 nm, and integrity was assessed by capillary electrophoresis utilizing the Agilent 2100 Bioanalyzer.  RNA-seq:  RNA was extracted in phenol: chloroform: isoamyl alcohol (PCA) followed by ethanol precipitation. DNA was enzymatically digested prior to a second round of PCA and ethanol precipitation. RNA purity was assessed by spectrophotometry at 260, 270, and 280 nm, and integrity was assessed by capillary electrophoresis utilizing the Agilent 2100 Bioanalyzer. All samples possessed RIN (RNA Integrity Number) values ≥9. ChIP-seq:  Library preparation was performed on ≥100 ng DNA using the Ovation Ultralow V2 DNA-Seq Library Preparation Kit (NuGEN) according to the manufacturer's instructions.  RNA-seq:  Library preparation was performed on ≥500 ng RNA using the Universal Plus mRNA-Seq Library Preparation Kit (NuGEN) according to the manufacturer's instructions.
GEO Accession
GSM5034047