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[…] e MIC to AR-13. Two representative AR-13r mutants were then passed in non-selective AR-13 free mTSB for 10 passes of an overnight culture. The stable AR-13r mutants were chosen for the subsequent studies including genomic sequencing, RNA sequencing, and assays regarding the mechanism of AR-13 resistance., Genomic DNA from wild-type LVS and its stable isogenic AR-13 resistant mutants were purified using the GenElute Bacterial Genomic kit (Sigma–Aldrich, Cat #PLN70). Samples were sequenced on the Illumina MiSeq instrument using paired-end 2 × 300 bp chemistry, generating more than 3 million read pairs per sample. Churchill () was run to identify any statistically supported variants, utilizing BWA_MEM v0.7.12 and the GCF_000009245.1 assembly of the F. tularensis LVS reference from NCBI for alignment and GATK HaplotypeCaller v3.5 for variant calling. Variants were annotated with SnpEff v4.1 against the GCA_000009245.1.26 annotation database., Similarly, the RNA from the wild-type LVS and stable isogenic AR-13 resistant mutants was purified from mid-log cultures in mTSB using the RNAeasy plus mini kit (Qiagen, Cat#74134). Using methods described previously (), after determining the quality of total RNA using Agilent 2100 bioanalyzer and RNA Nano Chip kit (Agilent Technologies, CA), rRNA was removed from 2 μg of RNA with Ribo-ZeroTM rRNA removal kit for Gram-Negative bacteria (Epicentre Biotechnologies, WI). To generate directional signal in RNA seq data, libraries were constructed from first strand cDNA using ScriptSeqTM v2 RNA-Seq library preparation kit (Epicentre […]

Pipeline specifications

Software tools BWA, GATK, SnpEff
Diseases Gram-Negative Bacterial Infections, Drug-Related Side Effects and Adverse Reactions
Chemicals Aminoglycosides