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. 2012 Sep 12;7(9):e44690. doi: 10.1371/journal.pone.0044690

Figure 8. Direct Amplification of open chromatin with phi29 polymerase: Translation of DC-PCR to genome-wide chromatin analysis.

Figure 8

CDKN2A PCR on KMS-12-PE supernatant after direct genome-wide phi29 amplification (A). Untreated KMS-12-PE cells or cells treated with DAC (1 µM for 3 d) were placed directly in phi29 reaction mix for 4.5 hrs at 30°C. After that, the reaction was centrifuged to pellet cell carcasses. Only the upper 50% of the supernatant was used for subsequent amplification of CDKN2A sites by conventional PCR. Supernatant from DAC treated cells yielded more CDKN2A products than vehicle treated cells. When DAC treated cells were incubated in reaction buffer without Phi29 polymerase no CDKN2A products could be amplified from supernatants by taq polymerase during conventional PCR. FOSB PCR on KMS-12-PE supernatants obtained from untreated cells or cells treated with 10-E-09 (10 µM for 6 h) after direct genome-wide phi29 amplification (B). The same procedure as above yielded FOSB PCR products from supernatants of 10-E-09 treated KMS-12-PE cells amplified by phi29 while vehicle treated cells or supernatants from 10-E-09 treated cells incubated in reaction buffer without phi29 yielded no FOSB regulatory region amplicons. Results are representative of three independent experiments.