ChIP-SEQ of ETV3-HA in 293T cells transfected with ETV3-HA and treated with PMA
Three 15-cm plates of HEK 293T cells were transfected (or not for negative control) with HA-tagged human ETV3 (20 µg plasmid, 60 µL FuGene HD in 0.5 mL PBS) for two days to a density of 25e6 cells per plate. Cells were treated for 20 minutes with U0126 (10 µM) and/or PMA (100 nM). A control experiment was conducted with non-transfected cells. Frozen pellets of formaldehyde-fixed cells were resuspended and lysed.  The cells were then sonicated using a Misonix Sonicator 3000 at 33W for twelve cycles of a 20 second pulse followed by a 60 second pause. Lysate was centrifuged at 14,000 rpm for 10 minutes at 4°C to remove impurities. The lysate was incubated with 50 µL of Dynal Protein G magnetic beads bound to 10 µg of antibody for 15 hours at 4°C.  The purified DNA was prepared for sequencing on a Beckman Coulter SPRI-TE following manufacturer’s instructions. The seq-prepped DNA was PCR amplified using Illumina primers for 18 cycles. Samples were sequenced using Illumina Solexa Genome Analyzers 2.0. Reads were mapped to the hg18 reference genome using the GERALD alignment software package.  Uniquely mapping reads were run through the MACS v1.4.0beta peak calling software using default parameters.
Massachusetts Institute of Technology
ETV3-HA ChIP-SEQ PMA-treated