RIP-PEN-SHAPE-MaP for 15.5K-interacted RNAs

RNA secondary structure is crucial for RNA mentalism, including transcription, splicing, translation, RNA-binding protein interaction as well as turnover. The kink-turn (K-turn) structure motif is is an RNA three-dimensional (3D) structure that exists in all three primary phylogenetic domains and plays vital roles in RNA metabolism. In order to investigate the K-turn secondary structure in vivo, we combined SHAPE-Map and our RIP-PEN-seq to profile the RNA structure of 15.5K-interacted RNAs. Overall design: The 15.5K-interacted RNAs were first isolated by 15.5K-specific antibody and followed by on-beads modification with SHAPE reagent NAI-N3 or DMSO (negative control). After RNA isolation and DNase I-treatment, the 15.5K-interacted RNAs were ligated to 3' and 5' randomized RNA adaptors. Then the ligated products were subjected to rRNA, snRNA and some high-abundance snoRNA depletion utilizing an RNase H-based strategy. Following the reverse transcription under SHAPE-MaP conditions, the libraries were amplified with Illumina small RNA indexed PCR primers.