GSM5229036: VCaP_AU_4h_H3K4me4 (HiChIP-seq); Homo sapiens; OTHER
Intact cells were crosslinked using with 3mM DSG and 1% formaldehyde and processed as described in the Dovetail MNase HiChIP kit (Cat No. 21007) protocol. HiChIP assay was performed on 5 million DMSO or AU-15330 treated (1uM for 4h) VCaP cells. Frozen cells were resuspended in 1X PBS and crosslinked with 3mM DSG and 1% formaldehyde. Washed cells were digested with 0.5 uL MNase in 100 uL of Nuclease digest buffer with MgCl2. Cells were lysed with 1X RIPA and clarified lysate (approximately 1400 ng) was used for ChIP. The antibody amount used per ChIP and vendor information are as follows: CTCF – 1.14 μg of Cell Signaling, Cat. 3418; H3K4me3 – 3.4 μg of Cell Signaling, Cat. 9751; H3K27ac – 0.4 μg of Cell Signaling, Cat. 8173. The Protein A/G bead pulldown, proximity ligation, and libraries were prepared as described in the Dovetail protocol (Dovetail™ HiChIP MNase Kit). Crosslinked DNA was fragmented using an MNase cocktail and immunoprecipitated using 5-10ug of target antibody from 5M cells per HiChIP.