Nascent RNA-sequencing
The cells at 70~80% confluency were treated with 1 mM EU for 15 minutes. The EU labelling was stopped by direct addition of TRI Reagent to cells. Total RNA was isolated and treated with DNase I. EU-labelled spike-in RNA were added to each sample for calibration. The RNA was then biotinylated with click chemistry and fragmented with NaOH. After RNA fragmentation, all biotinylated RNAs were pulled down onto Dynabeads MyOne Streptavidin C1 (Invitrogen) by immunoprecipitation. The 3-phosphate groups were removed using FastAP, the 3-RNA-adapter was ligated using T4 RNA ligase 2 (New England Biolabs). cDNA was synthesized through reverse transcription reaction using specific RT-Primer. Dynabeads MyOne Silane (Invitrogen) were used to enrich single cDNA. T4 RNA ligase 1 (New England Biolabs) was used to ligate the 5-DNA-adapter. The cDNA was purified for next-generation sequencing library construction.