Accedi

Ludwig-Maximilians-Universität München

2 ARTICLES PUBLISHED IN JoVE

Biology

Metabolic Labeling of Newly Transcribed RNA for High Resolution Gene Expression Profiling of RNA Synthesis, Processing and Decay in Cell Culture

Bernd Rädle^*1, Andrzej J. Rutkowski^*2, Zsolt Ruzsics¹, Caroline C. Friedel³, Ulrich H. Koszinowski¹, Lars Dölken²

¹Max von Pettenkofer Institute, ²Department of Medicine, University of Cambridge, ³Institute for Informatics, Ludwig-Maximilians-University Munich

Total cellular RNA provides a poor template for studying short-term changes in RNA synthesis and decay as well as the kinetics of RNA processing. Here, we describe metabolic labeling of newly transcribed RNA with 4-thiouridine followed by thiol-specific biotinylation and purification of newly transcribed RNA allowing to overcome these limitations.

Genetics

Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation

Kathrin Davari^*1, Johannes Lichti^*1, Caroline C. Friedel², Elke Glasmacher^1,3

¹Institute for Diabetes and Obesity (IDO), German Center for Diabetes Research (DZD), Helmholtz Zentrum München, ²Institute for Informatics, Ludwig-Maximilians-Universität München, ³Roche Pharma Research and Early Development, Large Molecule Research, Roche Innovation Center Penzberg

This protocol describes the combinatorial use of ChIP-seq, 4sU-seq, total RNA-seq, and ribosome profiling for cell lines and primary cells. It enables tracking changes in transcription-factor binding, de novo transcription, RNA processing, turnover and translation over time, and displaying the overall course of events in activated and/or rapidly changing cells.