RIL-seq of Klebsiella pneumoniae SGH10 grown in LB or DMEM medium
RIL-seq of Klebsiella pneumoniae SGH10 grown in LB or DMEM medium
Bacteria were grown in LB (Luria Bertani) medium and in DMEM (Dulbecco's Modified Eagle Medium) to exponential phase and cells were harvested at OD600 = 0.5. Cells were peleted by centrifugation, resuspended in PBS and subjected to UV crosslinking. Then bacteria were re-collected by centrifugation and flash frozen in liquid nitrogen Following mechanical lysis of the cells, Hfq with its bound RNAs was coimmunoprecipitated using anti-Flag antibody. Exposed regions of the RNAs were trimmed by RNases, the 50 and 30 ends of the RNAs were treated with T4 PNK, neighboring RNAs were ligated, the Hfq protein was digested, and RNA was then isolated using TRI-reagent  RNAtag-seq with mudifications detailed in Melamed et al, Nature Protocols 2018