Computational protocol: DNA polymerase κ-dependent DNA synthesis at stalled replication forks is important for CHK1 activation

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Protocol publication

[…] To knock down the POL K gene, cells were transfected with two independent siRNA () or Pol κ siRNA Smart Pool (Dharmacon) directed either the coding sequence (D-siκ) or the 3'UTR region (si-κ3′UTR). For control, siRNA against luciferase () was used. For siRNA transfection experiments, 1−1.5 × 105 cells were seeded 24 h before transfection with 100–300 nM siRNA using lipofectamine 2000 (Invitrogen). For vectors transfections, 2 μg of DNA was transfected with JetPrime reagent (Polyplus-Ozyme). Chromatin fractionation was performed as in with the following modifications: solution A (10 mM PIPES pH 7, 300 mM sucrose, 3 mM MgCl2, 150 mM NaCl, 1 mM EGTA, 1 mM DTT, 0.1%, Triton X-100, 0.5 μg/ml Pepstatine and 1X to 3X HaltTM protease/hosphatase inhibitors; Thermo Scientific) was used. Cells were left on ice during 5 min then spun 3 min at 7500, r.p.m. at 4°C. Pellets were incubated in solution A for 5 min on ice, spun again, and resuspended in solution A (Chromatin fraction). The supernatants from the two extraction steps were pooled (soluble fraction). Chromatin fractions were sonicated. Whole cell extracts were performed as previously described (). Blots were detected by ECL Western Blotting Substrate (Pierce) or ECL Plus Western blot detecting system (GE Healthcare). Where indicated, western blots were quantitated using ImageJ software. [...] Images acquisition of multiple random fields were carried out on a wide field Leica DMLA microscope equipped with × 63 oil immersion objective (PLAPO 1.4 Leica) using a Cool-Snap HQ CCD camera (Photometrics, Roper Scientific) driven by PM capture Pro6.0 or Metamorph software. Images were assembled with Adobe photoshop and Illustrator. When indicated in the figure legends, images of RPA were acquired using the × 100 NA 1.4 objective of a SP2 confocal microscope (Leica microsytems) using laser lines 488 and 633 nm for excitation of Alexa Fluor 488 and ToPro3 dyes. […]

Pipeline specifications

Software tools ImageJ, MetaMorph
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Xenopus laevis, Homo sapiens