Protocol for simultaneous profiling of transcription start sites and full-length transcripts from low-input samples...

Transcriptomic approaches such as cap analysis of gene expression (CAGE) enable the identification of transcription start sites (TSS) and the quantification of promoter activity. However, these techniques require large RNA input amounts and cannot profile transcript bodies simultaneously. Here, we present a protocol for characterizing full-length transcripts while simultaneously identifying the precise location of TSSs using Smart-seq+5', a low-input library preparation approach. We describe steps for RNA extraction with optional in vitro polyadenylation, reverse transcription and 5' capture, Tn5 tagmentation for transcript coverage, and computational analysis. Based on the widely adopted Smart-seq2, Smart-seq+5' offers improved sensitivity and can be employed to profile both polyadenylated and non-polyadenylated transcripts. For complete details on the use and execution of this protocol, please refer to Oomen et al.¹.