Protocol for RNA-seq library preparation from low-volume total RNA by RNA/cDNA hybrid tagmentation

RNA sequencing (RNA-seq) is a widely used and powerful technique for studying gene expression. Among the various protocols, SHERRY (sequencing hetero RNA-DNA-hybrid) profiles polyadenylated RNAs by direct tagging of RNA/DNA hybrids and offers a robust and economical way for gene expression quantification. Here, we present a detailed protocol for standard SHERRY library preparation from 200 ng of total RNA. We describe steps of RNA purification, reverse transcription, hybrid tagmentation, and library generation. We then detail procedures for sequencing and data analysis. For complete details on the use and execution of this protocol, please refer to Di et al.¹.