Computational protocol: AR-13, a Celecoxib Derivative, Directly Kills Francisella In Vitro and Aids Clearance and Mouse Survival In Vivo

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Protocol publication

[…] 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 Biotechnologies, WI). rRNA-depleted RNA (50 ng) was fragmented and reverse transcribed using random primers containing a 5′ tagging sequence, followed by 3′ end tagging with a terminal-tagging oligo to yield di-tagged, single-stranded cDNA. After magnetic-bead based purification, the di-tagged cDNA was amplified by limit-cycle PCR using primer pairs that anneal to tagging sequences have adaptor sequences required for sequencing cluster generation. The AMPure XP System (Beckman Coulter) was used to purify RNA-seq libraries. The Agilent 2200 TapeStation using High Sensitivity D1000 ScreenTape was used to determine the quality of libraries, which were then quantified using the Kappa SYBR®Fast qPCR kit (KAPA Biosystems, Inc., MA, United States). On average, 21 million paired-end 150 bp RNA-Seq reads were generated using the Illumina HiSeq 4000 platform for each sample (the range was 19 to 24 million). Each sample was aligned to the GCF_000009245.1 assembly of the F. tularensis LVS reference from NCBI using version 0.7.5a of BWAMEM. The GFF file provided with the GCF_000009245.1 assembly from NCBI was used to identify transcript features and raw coverage counts were calculated using HTSeq. After normalization of the RNA-Seq gene expression data a post-alignment statistical analyses was performed using custom analysis scripts written in R and DESeq2. Normalized read counts were used for comparisons of gene expression and associated statistical analysis of the different bacteria/conditions. Fold change values were displayed as test condition/control condition, where values less than one were expressed as the negative of its inverse (note that there will be no fold change values between -1 and 1, and that the fold changes of “1” and “-1” represent the same value). A false discovery rate of 10% (DESeq2 adjusted p-value <= 0.1) was used in determining transcripts that were significantly differentially expressed. […]

Pipeline specifications

Software tools BWA, GATK, SnpEff, HTSeq, DESeq2
Application RNA-seq analysis
Diseases Tularemia, Drug-Related Side Effects and Adverse Reactions
Chemicals Gentamicins