Rna clean concentrator columns

RNA was isolated from triplicate mid-log cultures for each strain using standard techniques. Briefly, cells from each culture were harvested by centrifugation, resuspended in 1 mL of TRIzol (Thermo Fisher), and disrupted by bead-beating. Chloroform was added to 25% of the total volume and total RNA was isolated using the Direct-zol RNA MiniPrep kit (Zymo Research). Samples were then treated with TURBO DNase (Ambion) for 1 hour to remove residual contaminating genomic DNA, followed by clean-up with RNA Clean & Concentrator columns (Zymo Research). RNA was quantified by Qubit RNA Assay (Thermo Fisher) and 25 ng of RNA was used as input in the NanoString nCounter assay (NanoString Technologies) with custom-designed probes to quantify gene expression. Target sequences are listed in S9 Table. Data were analyzed with nSolver version 4 (NanoString Technologies); raw NanoString counts were normalized to internal positive controls to correct for technical variation between assays, and normalized to a housekeeping gene (sigA) to correct for variation in RNA input amount. Background counts from no-input samples were subtracted.

Templates for gRNA transcription with T7 polymerase were generated by two-oligo PCR, using one oligo containing the T7 promoter and gene-specific sequence with overlap to a second reverse complement oligo containing the constant region of the gRNA backbone. An optimized gRNA constant region was used24 (link), except in Supplementary Fig. 13 where two constant regions are compared for one gRNA. As in previous work8 (link)25 (link), the bases at the first two positions in the gRNA were always substituted for guanine to accommodate preferences of T7 RNA polymerase. The product was purified with the E.Z.D.A. Cycle-Pure kit (Omega Bio-tek, Norcross, GA). All gRNAs were transcribed with the Megascript T7 kit (Thermo Fisher Scientific, Waltham, MA), using half-size (10 μl) reactions and DNase treated. Purification of the gRNA was either done with ammonium acetate/ethanol precipitation or with column purification, using RNA Clean & Concentrator columns (Zymo Research, Irvine, CA), if precise quantification of gRNA concentration was required (Fig. 2). For column-purified gRNAs, RNA concentration was determined using a Nanodrop spectrophotometer and all gRNAs were visualized with agarose gel electrophoresis. The random RNA control was concentration matched to gRNAs and similarly sized, as well as equivalently column purified.

mRNA was isolated as described previously^{21 (link)}. Briefly, cells from 25 ml of a culture grown in BHI broth (OD₆₀₀ of ~0.8) was mixed with 25 ml ice cold killing buffer (20 mM Tris-HCl pH 7.5, 5 mM MgCl₂, 20 mM NaN₃) and harvested by centrifugation after 5 min incubation on ice.
RNA extraction followed the protocol of Gertz et al.^{51 (link)} with modifications as described^{18 (link)}. 10 µg total RNA were digested with DNAse using the RNase-Free DNase Set (Qiagen) and then purified using RNA clean & concentrator columns (Zymo Research) for purification for RNA molecules longer than 200 nucleotides. RNA quality was assessed using Agilent Bioanalyzer RNA Nano chips. rRNA was depleted using the Ribo-Zero Bacteria Kit (Illumina), 2 µg purified RNA was treated with 10 µl Ribo-ZeroRemoval Solution and pelleted by ethanol precipitation. RNA concentrations were determined in a Qubit® fluorometer.