Azidonucleoside-incorporated RNA sequencing for new transcripts analysis under heat shock in E.coli

Cellular RNA levels result from the interplay of RNA transcription, processing, and degradation. Multiple complementary methods have been developed to dissect these tightly regulated processes. One of the methods exploits chemically active nucleoside analogs (i.e., noncanonical nucleosides) to metabolically label newly transcribed RNA. The labeled RNA are then chemically conjugated with fluorophores for imaging or affinity tags for enrichment and sequencing, which allow for studying transcription by separation of nascent RNA from the pre-existing populations. Here we used AzG to profile RNA dynamic changes under heat shock in E.coli. Overall design: E. coli grown under normal condition at 37 ? or under mildly heat shocked condition at 42 ? were metabolically labeled with AzG for 10 min, followed by extraction of total RNA. After reacting with DBCO-biotin, the newly transcribed RNA were isolated by streptavidin beads and subjected to RNA-seq.