Computational protocol: Escherichia coli O157:H7 Acid Sensitivity Correlates with Flocculation Phenotype during Nutrient Limitation

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Protocol publication

[…] One library for each strain (B201, B241, and B250) was prepared using the Ion Total RNA-Seq Kit v2 (Thermo Fisher Scientific) following the manufacturer's instructions with one exception: incubation time for RNA shearing was reduced to 2.5 min. The final cDNA library was quantified with a 2100 Bioanalyzer using a High Sensitivity DNA Chip (Agilent). Library enrichment was carried out with an Ion PGM™ Template OT2 200 kit and Ion OneTouch™ 2 system (Thermo Fisher Scientific). Each library was sequenced with an Ion 318 Chip using an Ion PGM 200 Sequencing kit (Thermo Fisher Scientific) on the Ion Personal Genome Machine (Thermo Fisher Scientific). RNA-Seq data were analyzed by ProteinCT Biotechnologies (Madison, WI, USA). Briefly, raw reads were mapped to the E. coli O157 B201 and B241 genome sequences (Baranzoni et al., ) using the Subjunc program from the Subread-1.4.6 package ( Raw gene counts were generated by FeatureCounts from the Subread package, and differential expression was calculated using the Limma package ( Transcriptomics data are available under NCBI BioProject number PRJNA381969. [...] Gene deletion mutants were confirmed through colony PCR. Briefly, recombinant and wild-type colonies were re-suspended in 30 μl of de-ionized water and incubated at 98°C for 5 min to lyse cells. PCR was performed with Platinum PCR Supermix High Fidelity recombinant and wild-type DNA templates (2 μl), linear cassette primers, and sequencing primers. PCR products were visualized by agarose gel electrophoresis, ran at 65V for 100 min, and confirmed by band size by comparison with standards (accession number CP015020 and CP015023 for B201 and B241, respectively). Additionally, gene deletion mutants and wild-type target genes were confirmed through PCR product sequencing by Eton Biosciences, Inc. (Durham, NC). PCR product chromatographs were analyzed through FinchTV software (Perkin Elmer, Waltham, MA) and the corresponding sequences were aligned with B201 (accession number CP015020) and B241 (accession number CP015023) sequences through GenBank Blastn software (National Center for Biotechnology Information, Bethesda, MD; Baranzoni et al., ). […]

Pipeline specifications

Software tools FinchTV, BLASTN
Application Sanger sequencing
Organisms Escherichia coli
Chemicals Acetic Acid