ZDB488 replicate 3
RNA was extracted from frozen exponential phase cell pellets using the RNASnap protocol (Stead et al. 2012). The resulting supernatants were purified using Zymo Clean & Concentrator-25 columns incorporating the on-column DNase treatment step (Zymo Research). The integrity of purified RNA was assessed using a TapeStation (Agilent). Then, ribosomal RNA was depleted using the Gram-negative bacteria RiboZero rRNA Removal Kit (Epicentre), and the final eluate was used as input for strand-specific RNA-seq library construction using the NEBNext RNA Library Prep Kit (New England Biolabs). Libraries were fractionated on 4% agarose E-gels (Invitrogen), and amplicons ranging from 0.28 kb were extracted and purified using the Zymoclean Gel DNA Recovery Kit (Zymo Research), quantified using a Qubit 2.0 fluorometer (Life Technologies), and stored at 80C prior to sequencing.