Optimization and testing the microplate-based MeDIP. HeLa cells were treated with 200 nM of DAC for 3 days and 300 nM of TSA for the last 24 hours. Media containing fresh drugs was replaced daily. Control cells (vehicle) treated identically with drug solvents (PBS and 100% ethanol). Cells were trypsinized and washed with PBS. For MeDIP input DNA was deproteinized using proteinase K, sheared by ultrasound (Bioruptor), RNase treated and denatured. Assays were done using in-lab made protein A-coated polypropylene 96-well plates. A, MeDIP was tested either when anti-5mC antibody (Diagenode) was first attached to wells and then DNA was added (No) or the DNA was preincubated (Yes) with the antibody first and then the mixture was added to protein A-coated 96-well plates and binding was done in ultrasonic bath. After the binding step, wells were washed, DNA was eluted and used in real-time PCR using primers to ALU and LINE elements as wells as the SFRP1 gene. B, MeDIP was done with the preincubation step using monoclonal anti-5mC antibodies from either Diagenode (5mC-D) or Aviva (5mC-A) companies. Monoclonal anti-Flag tag antibody was used as control. Results are expressed as % Input, mean ± SEM of three independent biological replicates.
Yu et al. BMC Molecular Biology 2011 12:49 doi:10.1186/1471-2199-12-49