Live-cell analysis of endogenous GFP-RPB1 uncovers rapid turnover of initiating and promoter-paused RNA Polymerase II.

Generation of GFP-RPB1 KI

Cells and Cell Culture. KI cells were generated from Sv40-immortalized MRC-5 fibroblasts, as described here ( 19 ). Detailed protocol can be found in SI Materials and Methods . Cells were cultured in a 1:1 mixture of Ham’s F-10 and DMEM (Gibco) supplemented with antibiotics and 10% FCS, at 37 °C; 20% O 2 , and 5% CO 2 in a humidified incubator. Immunofluorescence and EU Incorporation. Click it-based EU incorporation was performed as described previously ( 55 ). A detailed protocol is in SI Materials and Methods . Immunofluorescence was performed as described in SI Materials and Methods . Transcription inihibitors, antibodies, and respective concentrations are listed in Dataset S2 . Cellular Fractionation, Western Blot Analysis. The fractionation procedure was adapted from ref. 56 and is described in detail in SI Materials and Methods . Fractions were separated on a 6% SDS Page gel, blotted overnight at 40 V. Blots were blocked with 1.5% BSA in PBS and stained with primary antibodies listed in Dataset S2 . Secondary antibodies were coupled to IRDyes (LiCor) and imaged with an Odyssey CLx infrared scanner (LiCor). Live-Cell Confocal Microscopy and Quantification of Nuclear Pol II. Live-cell imaging was performed on a Leica SP5 confocal laser scanning microscope with a HCX PL APO CS 63×, 1.40-NA oil-immersion lens. Images were recorded with a 488-nm Argon laser and a 500- to 600-nm bandpass filter. For the detailed RPB1 FRAP procedure, please see SI Materials and Methods . For Pol II concentration measurements eGFP (Biovision) was dissolved in PBS, serially diluted in culture medium, and the fluorescence intensity was measured alongside nuclear GFP-RPB1 fluorescence. For a detailed procedure, see SI Materials and Methods . MC Modeling. Experimental FRAP curves were fit by least squares, to a large set of computer simulation-generated FRAP curves that were computed based on a model that simulates diffusion of molecules (here Pol II), and binding to and releasing from immobile elements, (here chromatin) in an ellipsoidal volume (here the nucleus). For details, see SI Materials and Methods . Detailed protocols for RNA-Seq data analysis, Chromatin immunoprecipitation, siRNA transfection, and RT-PCR can be found in SI Materials and Methods .