MS2-Affinity Purification Coupled with RNA Sequencing in Gram-Positive Bacteria

Although small regulatory RNAs (sRNAs) are widespread among the bacterial domain of life, functions many them remain poorly characterized notably due to difficulty identifying their mRNA targets. Here, we described a modified protocol MS2-Affinity Purification coupled with RNA Sequencing (MAPS) technology, aiming reveal all partners specific sRNA in vivo. Broadly, MS2 aptamer is fused 5’ extremity interest. This construct then expressed vivo, allowing MS2-sRNA interact its cellular partners. After harvesting, cells mechanically lysed. The crude extract loaded into an amylose-based chromatography column previously coated protein maltose binding protein. enables capture and interacting RNAs. elution, co-purified identified by high-throughput sequencing subsequent bioinformatic analysis. following has been implemented Gram-positive human pathogen Staphylococcus aureus is, principle, transposable any bacteria. To sum up, MAPS technology constitutes efficient method deeply explore network particular sRNA, offering snapshot whole targetome. However, it important keep mind that putative targets still need be validated complementary experimental approaches.