Comparative analysis of nascent RNA sequencing methods in Arabidopsis

High throughput detection of nascent RNA is critical for studies of transcription and much more challenging than that of mRNA. Recently, several massively parallel nascent RNA sequencing methods were established in eukaryotic cells. Here, we systematically compared four different methods in Arabidopsis with same nuclei preparation using similar cDNA library preparation protocols. Reproducibility, sensitivity and accuracy in detecting nascent transcripts, experimental procedures, and costs were analyzed, which revealed the strengths and weaknesses for each method. We found that pNET-seq and GRO-seq performed best in detecting transcribing RNA polymerase II, CB RNA-seq is easy and costly effective, and 3'CB RNA-seq is sensitive for transcription coupled splicing.