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Complete transcriptome of bir-2 knockdown C. elegans nematodes with and without exposure to pathogenic bacteria Enterococcus faecalis.

ABSTRACT: Purpose: The goals of this study are to examine the role of C. elegans gene bir-2 in innate immunity. The method is to compare differences in gene expression of transcriptomes of C. elegans in the presence and absence of a bir-2 knockdown and with and without pathogenic bacteria. Results: We mapped about 4 million sequence paired-end reads of 251 nucleotide length for each of the 16 samples, trimmed the reads with Trimmomatic (v.0.38), mapped to the WBGene C. elegans genome (build WBcel235.99) using HISAT2 (v.2.1.0), assembled and merged transcripts with StringTie. SamTools were used to convert the BAM files. Differential expression was evaluated using DESEQ2 in R. Approximately 4% of the transcripts showed differential expression between the WT and bir-2 knockdown, with a fold change ?2 and p value <0.05.!Series_summary = Methods: C. elegans were grown, in quadruplicate, exposed to HT115 bacteria expressing RNAi for bir-2 with and without E. faecalis exposure. Total RNA was purified and Illumina poly-A selection, library construction (KAPA RNA Hyperprep) and deep sequencing, using Illumina HiSeq2000 at UNH Hubbard Genome Center. The sequence reads were passed through FASTQC and Trimmomatic, and were analyzed at the transcript isoform level. Conclusions: Our study represents the first detailed analysis of retinal transcriptomes, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions.

ORGANISM(S): Caenorhabditis elegans

PROVIDER: GSE184086 | GEO | 2022/12/31

REPOSITORIES: GEO

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Project description:Purpose: The goal of this study was to use RNA-seq to define the Klebsiella pneumoniae transcriptome recorded under 5 different experimental conditions, and to identify signature genes of each condition by comparing global transcriptional profiles. Methods: mRNA profiles were generated for Klebsiella pneumoniae CH1034 clinical isolate, in triplicate, by deep sequencing. Total RNAs were harvested from bacteria cultured at 37Â°C in M63B1 minimal media under different conditions: (i) planktonic aerobic condition at OD 620nm=0.250 (exponential growth-phase), (ii) overnight planktonic aerobic condition (stationnary growth-phase), (iii) biofilm in a flow-cell chamber after 7 hours of incubation (7-hours old biofilm), (iv) biofilm in a flow-cell chamber after 13 hours of incubation (13-hours old biofilm), (v) bacteria self-dispersed from biofilm recovered in the flow-cell effluent (biofilm-dispersed bacteria). Ribosomal RNAs were removed using the Bacteria Ribo-Zero Magnetic kit (Epicentre Biotechnologies). Libraries were prepared using the TruSeq Stranded mRNA Sample Preparation kit (Illumina), and 50bp single-reads were obtained by HiSeq 2000 (Illumina).The sequence reads that passed FastQC quality filters were mapped to the CH1034 genome using BurrowsâWheeler Aligner (BWA) (0.7.12-r1039 version). The transcript levels were determined using HTSeq-count (0.6.1p1 version) with union mode followed by DESeq (1.16.0 version) analysis. qRTâPCR validation was performed using SYBR Green assays. Results: We found that each condition has a specific transcriptional profile, and we identify 4 robust signature genes for each. Conclusion: Our study represents the first detailed analysis of K. pneumoniae transcriptomes under different experimental conditions generated by RNA-seq technology. The data reported here should permit the dissection of complex biologic functions involved in the transition between the sessile and planktonic modes of growth. Determination of the transcriptional profiling of Klebsiella pneumoniae under 5 different experimental conditions. mRNA profiles were generated for bacteria under exponential planktonic growth-phase, stationary planktonic growth-phase, 7 hours-old biofilm, 13 hours-old biofilm and biofilm-dispersed modes, each in three biological replicates, by deep sequencing using Illumina HiSeq

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Complete transcriptome of bir-2 knockdown C. elegans nematodes with and without exposure to pathogenic bacteria Enterococcus faecalis.

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