Comparative analysis of AMOUR and other strategies for profiling surface RNAs
Conventionally limited to proteins, lipids, and polysaccharides, cell membranes have recently revealed the presence of protein-coding RNAs and glycan small RNAs on the surface of mouse and human cells. Despite glycan-RNA's prevalent presence on the cell exterior, a comprehensive method for accurately mapping all surface RNAs, particularly for rare cell populations has been lacking, leaving their physiological roles largely unexplored. We developed AMOUR, an RNA capture technique coupled with T7-based linear amplification, enabling precise profiling of surface RNAs in mammalian cells while preserving plasma membrane integrity.To ensure both sensitivity and specificity, we developed two supplementary strategies: WGA-Pd and RT-Amp, for surface RNA profiling alongside AMOUR. Overall design: To ensure both sensitivity and specificity, we developed two supplementary strategies for surface RNA profiling alongside AMOUR. The first employs a wheat germ agglutinin pull-down assay (WGA-Pd), utilizing WGA to capture modified plasma membranes rich in N-acetylglucosamine and N-acetylneuraminic acid (sialic acid), followed by surface RNA extraction. The second involves a modified AMOUR method, substituting T7 RNA polymerase with reverse transcriptase during surface RNA capture. We also compared the surface RNA profiles post-protein deprivation with trypsin treatment (WGA-T).