Primer and fluorescently labeled internal probe sequences

RT-qPCR was used to measure enrichment of miR-21 and miR-181b promoter sequences in the ChIPed DNA using primer and fluorescently labeled internal probe sequences specific to each promoter (Integrated DNA Technologies, Coralville, IA). The primers and their coordinates are shown in Supplementary Table 1. Samples were analysed in duplicates. The PCR reaction (25 μl) contained 5 μl ChIP DNA, 12.5 μl of 2x TaqMan real-time PCR Master Mix containing DNA polymerase and dNTPs (Applied Biosystem, Foster City, CA) and 100 nM of primer/probe mix (Integrated DNA Technologies). The PCR conditions were: 2 min at 50°C, 10 min at 95°C, followed by 40 cycles with 15s at 95°C and 1 min at 60°C (combined annealing and extension), using the Bio-Rad CFX96 Real-Time System. Relative enrichment of DNA sequences was calculated by normalizing averaged cycle threshold (Ct) values to the input DNA values. These values are presented as fold change relative to DNA from the IgG-immunoprecipitated samples (set at 1-fold).