Chromatin was prelabeled with [14C]thymidine (0.5 pCi/ml) for 24 hours before siRNA transfection. Thirty hours after transfection, nascent chromatin was labeled with [3H]thymidine (25 nCi/ml). To compensate for lower replication rate in TLK2- and FLASH-depleted cells, labeling times were adjusted to obtain similar [3H]thymidine incorporation (10, 15, and 30 min for siRNA control, siTLK2, and siFLASH, respectively). Cells were lysed in hypotonic buffer [10 mM tris (pH 7.4), 2.5 mM MgCl2, and 0.5% NP-40, protease and phosphatase inhibitors], and nuclei were resuspended in digestion buffer [10 mM tris (pH 7.4), 10 mM NaCl, 5 mM MgCl2, and 2 mM CaCl2, protease and phosphatase inhibitors] and subjected to digestion with MNase (0.01 U/μl) (Worthington Biochemical Co.) at 37°C. Undigested chromatin was pelleted by centrifugation at 1500g for 2 min, and 14C and 3H activity in supernatant and undigested chromatin were measured with a liquid scintillation counter (LS 6500, Beckman Coulter). Readings were corrected for 14C bleed through into the 3H channel. Graphs show one representative experiment of three biological replicates.

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