GSM7027242: BW25113 nudC KO, replicate 3; Escherichia coli; RNA-Seq
Total RNA was extracted using RNazol RT (RN 190) (Molecular Research Center, Inc.) according to the manufacturer's recommendations. rRNAs were depleted using RiboMinus Bacteria 2.0 Transcriptome Isolation Kit (Invitrogen). The remaining RNA was fragmented into approximately 100-400 nt long pieces in ZnCl2 buffer and dephosphorylated using T4 PNK (Thermo Fisher Scientific). 140 ng of fragmented and dephosphorylated RNA was then used for RNA library preparation according to the NEXTflex Small RNA-seq Kit v3 (PerkinElmer) with minor modifications: at Steps E and G incubation at 42°C and 72°C was extended to 60 min, at Step G 9 PCR cycles were performed and Steps F and H1 were carried out as described in an alternative protocol for Preparing Libraries without Size Selection. RNA libraries up to 500 nt in length were purified from PAA gel as outlined in Step H2.