Figure 6: In vitro transcription through the Cga +1 nucleosome.
From: H2A.Z controls the stability and mobility of nucleosomes to regulate expression of the LH genes
(a) Description of experimental procedure for in vitro transcription experiments. (b) To rule out the possibility of different transcription initiation efficiency between the samples, we exploited the restriction enzyme site of NcoI, located at +25 on the transcription template, relative to the TSS. RNAP should protect DNA from NcoI digestion if it is stalled at +26. To quantify the degree of protection, DNA was purified and subjected to qPCR with specific primers spanning the transcription construct. (c) Naked and nucleosomal Cga +1 DNA were ligated to DNA containing a T7A1 promoter and subjected to in vitro transcription for 1 min. The RNA was purified, DNase I treated and reverse transcribed. RNA levels were quantified using qPCR with specific primers. Data shown as mean±s.e.m., n=9–10; P=0.01, 0.04 and 5 × 10−4, two-sample Student’s t-test. *P<0.05, ***P<0.001. nts, nucleotides.
