Simultaneous labeling of intracellular RNAs with BrU and 4sU. (A) Schematic illustration of the labeling procedure. HeLa cells were precultured in medium containing 150 μM BrU, and then the medium was changed to one containing 200 μM 4sU. Black, blue, and red lines indicate kinetics of expression of total RNAs, BrU-labeled RNAs, and 4sU-labeled RNAs, respectively. (B) Preparation and quantification of BrU-labeled and 4sU-labeled RNAs. Total RNAs were isolated and purified from labeled cells in time series and then divided into two samples. One was immunoprecipitated using an anti-BrdU antibody, and the cDNA was reverse-transcribed to be provided to QuantSeq (IP-RNAs). The other was alkylated using IAA, and the cDNA was reverse-transcribed to be provided to QuantSeq (Alkyl-RNAs). Because alkylated 4sU pairs with guanine (G) instead of adenine (A) during reverse transcription, the 4sU-labeled RNAs were identified as those including T > C conversions.
