This assay was performed as described (14), with some modifications. Briefly, EX2 cells were prepared as described for the ChIP protocol and, after TA incubation, collected by trypsinization. Cells were resuspended in ice-cold RSGB40 buffer [10 mM tris (pH 7.4), 10 mM NaCl, 3 mM MgCl2, 10% glycerol, and 0.5% NP-40] and centrifuged (3 min, 7000 rpm at 4°C) to isolate nuclei. Nuclei were washed with digestion buffer [10 mM tris (pH 7.4), 15 mM NaCl, 60 mM KCl, and 1 mM CaCl2], centrifuged (3 min, 7000 rpm at 4°C), and resuspended in digestion buffer. One-half was digested with 10 U of MNase (Fermentas) for 30 min at 37°C before incubation with stop buffer [100 mM EDTA and proteinase K (20 μg/ml)] for 30 min at 55°C, and another half was treated with buffer alone (nondigested). Mononucleosome-sized DNA obtained after 30-min digestion and nondigested DNA were used after standard phenol-chloroform purification as templates for RT-qPCR. The amount of MNase-resistant DNA at each gene specific region was estimated as follows: 2^(Ct nondigested – Ct MNase digested), where Ct t0 and Ct t30 are the mean threshold cycles of RT-qPCR done in duplicate on DNA samples from nondigested (t0) and 30-min MNase-digested (t30) samples, respectively. Data were normalized against the MNase values were obtained in control cells (i.e., without TA). Primer sequences are shown in table S1.

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