Extended Data Fig. 8: Purification and sequence analysis of small guide RNAs associated with NbaSPARDA in E. coli.
a, Small RNAs associated with WT SPARDA at different steps of the standard purification protocol (Ni-chelating, heparin and MonoQ columns). Most gRNAs are removed after heparin chromatography. The OD260/280 ratio of the final sample is 0.55. b, Small RNAs associated with WT and mutant SPARDA variants at different steps of purification using a modified protocol, in which EDTA was omitted from the buffers during heparin and anion-exchange chromatography. Significant amounts of gRNAs remain associated within the WT and CD complexes (but not the MID mutant) through all purification steps, indicative of stable gRNA binding. The OD260/280 ratios of the final WT, MID and CD samples are 0.98, 0.53 and 0.82, respectively. Panels a,b show representative gels from two independent experiments, which produced similar results. c, Comparison of the activities of WT SPARDA purified by different protocols. Representative gels from 3 independent experiments. SPARDA samples containing endogenous gRNAs (OD260/280 = 0.98, right) have a lower nuclease activity in comparison with guide-free SPARDA (OD260/280 = 0.55, left), when activated by specific gRNA (20 nt) and tDNA (50 nt). Results d, Correlation between the abundance of gRNAs and the RNA transcriptome of E. coli strains expressing NbaSPARDA from pBAD and containing pACYC184 (three individual replicate experiments for Fig. 3h). Plasmid genes are shown in violet and blue (pBAD and pACYC, respectively). Regulatory antisense RNAs, prophage genes, and IS elements are shown in orange, pink and turquoise, respectively. e, Analysis of the distribution NbaSPARDA-associated gRNAs and long RNAs along plasmid sequences, pBAD (top) and pACYC184 (bottom). Short gRNAs and total RNA were isolated from E. coli containing pBAD_NbaSPARDA and pACYC184 at 5 h after induction of SPARDA, sequenced and mapped to the plasmids. The amounts of short gRNAs and long RNA transcripts are shown in RPKM (reads per kilobase per million aligned reads in the library) for each DNA strand and plotted for each plasmid region (left and middle panels, respectively). The ratio of short to long RNAs is shown on the right panels. Means and standard deviations from three replicate experiments. The map of each plasmid is shown above the plots. f, Distribution of various types of transcripts among the genes enriched or depleted for gRNAs. For all E. coli genes, the ratios of small gRNAs to long RNA transcripts were calculated, and the genes were sorted by this ratio in descending order (from left to right). The distributions of coding, noncoding and regulatory antisense transcripts among the top and bottom 100 genes are shown in circular diagrams. The list of top enriched genes (with the highest ratio of short to long RNAs) is shown below the diagram. g, Distribution of gRNAs and long RNAs along the E. coli chromosome. Short gRNAs and total RNA isolated from E. coli cells containing pBAD_NbaSPARDA and pACYC184 were mapped to the MG1655 reference genome (GenBank ID GCA_000005845.2), the amounts of short gRNAs and long RNA transcripts were expressed in RPKM for each E. coli gene and their ratios were calculated. Only reads corresponding to the sense strand of each gene were counted. The sequences corresponding to rRNA were omitted from analysis. Means from three replicate experiments.
