H3K4me3 ChIP was performed in T47D cells with 2.5×106 cells/IP, as previously described (Carroll et al., 2005 (link)). In brief, cells were treated as indicated. Cells were crosslinked, lysed in SDS lysis buffer, and sonicated (Covaris). H3K4me3 antibody (3ug) was added to sonicated lysates per o/n IP. Protein A beads (Novex) were incubated with IP for 6h. Chromatin was eluted and reverse crosslinked o/n and DNA was recovered with RNAse A incubation. Glycogen and Proteinase K were added and incubated at 62°C. DNA was purified and eluted in Low TE. KDM5A and HA ChIPs were performed using the Sarkosyl method (Lee et al., 2006 (link)) with 2.5×108 cells/ChIP and KDM5A #1416 antibody (10ug) (Dr. William Kaelin) or HA ab9110 (5ug) (abcam), respectively.
For samples that were Illumina sequenced, ChIP eluate was purified (Ampure) and indices added (ThruPLEX-FD Prep kit, Rubicon Genomics). Samples were then size selected (Pippin Prep, Sage Science) and purified (Ampure). DNA quality and quantity was measured using a Fragment Bioanalyzer and samples submitted to the Center for Functional Cancer Epigenetics for sequencing on a Next-Seq Illumina platform.
For samples that were Illumina sequenced, ChIP eluate was purified (Ampure) and indices added (ThruPLEX-FD Prep kit, Rubicon Genomics). Samples were then size selected (Pippin Prep, Sage Science) and purified (Ampure). DNA quality and quantity was measured using a Fragment Bioanalyzer and samples submitted to the Center for Functional Cancer Epigenetics for sequencing on a Next-Seq Illumina platform.