Simultaneous measurement of transcription and degradation rates by using double-labeling of RNA with BrU and 4sU

The purpose of this project is to simultaneously measure transcription and degradation rates on HeLa cells in steady state. First, HeLa cells are pre-cultured in a medium including bromouridine (BrU), and subsequently the medium is replaced with which including 4-thiouridine (4sU). Total RNA is obtained from the double-labeled HeLa in time series. For the converted samples (Converted sample from double-labeled HeLa at XXX), the total RNA are then alkylated by IAA causing T > C conversions to extract 4sU-labeld (= nascent) RNA. For immunoprecipitated samples (IP sample from double-labeled HeLa XXX), the total RNA are then immunoprecipitated using an anti-BrU antibody to extract the BrU-labeld RNA. The 4sU- and BrU-labeld samples are sequenced using Illumina Hiseq3000 in 100bp paired end (Note that read2 data are polyA sequences due to library preparation). The sequence data can be used to quantify the nascent and decomposed RNA at each time point to estimate transcription and degradation rates.