Dataset S1. Differentially Expressed Genes in E. cloacae upon interaction with Noroviruses as determined by RNA-seq

Published: 13 April 2021| Version 1 | DOI: 10.17632/nzmbn76h2h.1
Contributor:
Melissa Jones

Description

Many enteric viruses have been shown to bind to commensal bacteria, which may facilitate enhancement of viral infection. Given the ability of these bacteria to rapidly respond to external stimuli, we hypothesized the viral binding would induce changes in bacterial gene expression. These data demonstrate that binding of both murine and human noroviruses to E. cloacae induces global changes in gene expression by the commensal bacterium.

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E. cloacae was grown until it reached the stationary phase, and the bacterial pellet was washed twice with 1X PBS. The bacterial cell count was adjusted with PBS to a final concentration of 108 cells/ml. Cells were inoculated with either MNV (0.1 MOI), HuNoV VLPs (0.1 µg/ml), silver nanoparticle (AgNP; equivalent volume to that of virus), or PBS. Only. The mixtures were incubated for 1 h at 37°C with constant, gentle mixing. Prior to RNA extraction, 500 µl of RNAlater (Ambion) was added to the 1ml aliquots of fresh attachment assay samples. Extraction for E. cloacae and B. thetaiotaomicron was performed using the Zymo RNA MiniPrep kit according to manufacturer’s instructions. RNA was eluted in a volume of 51 µl nuclease-free water. Following the extraction, genomic DNA was removed using the Turbo DNA-free kit (Ambion) according to the “Rigorous DNase treatment” protocol in the kit instructions. RNA Sample QC, library preparations and sequencing reactions were conducted at GENEWIZ, LLC. 16 RNA samples were quantified using Qubit 2.0 Fluorometer (Life Technologies) and RNA integrity was checked with 4200 TapeStation (Agilent Technologies). An rRNA depletion was performed using Ribozero rRNA Removal Kit (Illumina). RNA sequencing library preparation used NEB Next Ultra RNA Library Prep Kit for Illumina by following the manufacturer’s recommendations. Briefly, enriched RNAs were fragmented for 15 minutes at 94°C. First strand and second strand cDNA were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3’ends, and universal adapter was ligated to cDNA fragments, followed by index addition and library enrichment with limited cycle PCR. Sequencing libraries were validated using the Agilent Tapestation 4200 (Agilent Technologies), and quantified by using Qubit 2.0 Fluorometer (Invitrogen) as well as by quantitative PCR (Applied Biosystems). The sequencing libraries were multiplexed and clustered on one lane of a flowcell and loaded on the Illumina HiSeq instrument according to manufacturer’s instructions. The samples were sequenced using a 2x150 Paired End (PE) configuration. Image analysis and base calling were conducted by the HiSeq Control Software (HCS). Raw sequence data (.bcl files) generated from Illumina HiSeq was converted into FASTQ files and de-multiplexed using Illumina's bcl2fastq 2.17 software. One mismatch was allowed for index sequence identification.