GSE174333
MdaB and NfrA, two novel reductases important in the survival and persistence of the major enteropathogen Campylobacter jejuni
Methods: RNA-Seq was used to identify differentially expressed genes between wild type strains, ?rrpA  and ?rrpB mutants at mid-log (~6 hrs) of growth. 11168H wild type, 11168H?rrpA, 11168H?rrpB and 11168H?rrpAB were plated out on BA plates and incubated at 37°C under microaerobic conditions for 6 hrs. RNA was extracted from the pelleted bacteria cells using PureLink™ RNA Mini Kit (Invitrogen), following manufactures protocol. RNA was isolated from five independent experiments performed on different days Ribosomal RNA was depleted using Ribominus (Invitrogen).Libraries were prepared using TruSeq® Stranded mRNA (Illumina).The paired-end reads were trimmed and filtered using Sickle v1.200  by using a sliding window approach and trimming the reads where the average base quality drops below 20. Only the reads that were above 10 bp length were kept after trimming. Bowtie2 was used to map the reads against the reference sequences; C. jejuni strains 11168H assembly GCA_900117385.1. This generated the mapping in SAM format which were converted to BAM format using Samtools  and were index sorted. We used gffread from cufflinks suite to convert annotations from GFF to GTF format. These were used in Bedtools (with multicov-bams option)with the mapped reads to generate transcript counts per samples. A shell utility by the authorswas then used to collate all these transcripts into a n=20 samples x p=1,628 transcripts for 11168H strain. Statistical analysis was performed in R using the combined data generated from the bioinformatics as well as meta data associated with the study (multifactorial design). We used DESeq DataSet from Matrix()function fromDESeq2  package with the adjusted p-value significance cut-off of 0.05 and log fold change cut-off of 1.5. This function uses negative binomial GLM to obtain maximum likelihood estimates for the transcripts log fold change between the two conditions. Overall design: Transcriptome profiles of Campylobacter jejuni 11168H and its ?rrpA, ?rrpB and ?rrpAB mutants.