Knockdown specificity of Mobile-CRISPRi system targeting chromosomal mRFP

The Mobile CRISPRi system with and without mRFP-targeting sgRNA was engineered into Pseudomonas aeruginosa PA14 strain with chromosomally encoded mRFP. RNA was isolated from these strains, and the corresponding cDNA library was synthesized and sequenced in 150 bp paired-end reads. Approximately 1,000,000 reads were collected for each of the two samples, with ~94% alignment to PA14 WT by Bowtie254, and transcripts were counted with HTSeq55. Only genes with a non-normalized read count greater than 1 in both samples were included in analysis, with a coverage of 1286 genes (~20% genome). This data shows that the Mobile CRISPRi system is selective for sgRNA-guided knockdown of mRFP. Overall design: The RNA concentration for each sample was determined with a NanoDrop spectrophotometer (Thermo Fisher Scientific). 10 ng total RNA of each sample was fragmented for 6 min, cDNA libraries were prepared using “NEBNext Ultra RNA Library Prep Kit for Illumina” (NEB#E7770S). Libraries were sequenced in collaboration with the Chan Zuckerberg Biohub in San Francisco on an Illumina MiSeq in 150 bp paired-end runs. Approximately 1,000,000 reads were collected for each of the two samples, with ~94% alignment to PA14 WT by Bowtie254, and transcripts were counted with HTSeq55. Only genes with a non-normalized read count greater than 1 in both samples were included in analysis, with a coverage of 1286 genes (~20% genome).