NPE supports regulated and promoter-dependent transcription.
A, pActin schematic. Sequence elements are shown relative to the transcription start site (+1). “Control” and “Promoter” primer pair locations are indicated. B, pActin was incubated at 10 ng/μl in NPE supplemented with buffer or α-amanitin. RNA was isolated at the indicated time points and quantified by RT-qPCR. C, different concentrations of pActin or ΔPromoter plasmid were incubated in NPE for 120 min. RNA was isolated and quantified by RT-qPCR using the Promoter primers. D, transcription from (C) was normalized based on starting plasmid concentration. E, pActin or ΔPromoter plasmid were incubated in NPE at 25 ng/μl. At 30 min, DNA-bound protein was analyzed by ChIP with the indicated antibodies. F, at 120 min, RNA was isolated from the reactions in (E) and quantified by RT-qPCR using the Promoter primers. Error bars represent ± 1 S.D. See Fig. S2 for experimental replicates.