GSM4041278: Planktonic_XL+_Rep1; Pseudomonas aeruginosa; RIP-Seq
Bacteria were lysed and the FLAG-tagged Hfq was immunoprecipitated using a monoclonal anti-FLAG antibody (Sigma, M8823). The samples were treated with benzonase nuclease (Sigma, E1014), calf intestine phosphatase (NEB, M0290), and polynucleotide kinase (Thermo, EK0032) in the presence of radioactive gamma-ATP. Samples were separated with SDS-PAGE and followed by transfering to nitrocellulose membranes. Radioactively labelled RNA-protein complexes were eluted from membranes and treated with Proteinase K. RNA was purified with phenol:chloroform extraction and ethanol precipitation. Purified RNA was used as input for library preparation using the NEBNext Small RNA Library kit according to the manufacturer's instructions.