Illumina HiSeq 2500 paired end sequencing; RNA-seq analysis of Helicobacter cinaedi ATCC BAA-847 under microaerobic and aerobic culture conditions
RNA-seq analysis of Helicobacter cinaedi ATCC BAA-847 under microaerobic and aerobic culture conditions
Following an initial inoculum of OD600 = 0.01, Helicobacter cinaedi ATCC BAA-847 was propagated at 37°C under the following mono-culture (bacteria) and co-culture (bacteria + epithelial monolayer) conditions: [1] monophasic (liquid alone) F12 medium, H2-supplemented microaerobic atmosphere (n=4); [2] biphasic (liquid over agar) F12 medium, H2-supplemented microaerobic atmosphere (n=4); [3] biphasic F12 medium, H2-free/CO2-supplemented microaerobic atmosphere (n=4); [4] F12 medium over Caco-2 monolayer, H2-supplemented microaerobic atmosphere (n=5); [5] F12 medium over Caco-2 monolayer, CO2-supplemented aerobic atmosphere (n=5); [6] monophasic F12 medium + 10 mM L-lactate, H2-supplemented microaerobic atmosphere (n=4); [7] monophasic F12 medium + 10 mM L-lactate, CO2-supplemented aerobic atmosphere (n=4); [8] monophasic Mueller Hinton broth, H2-supplemented microaerobic atmosphere (n=4); [9] biphasic F12 medium + 10% normal human serum (commercially acquired), H2-supplemented microaerobic atmosphere (n=5). After 24 hours of growth, total RNA was extracted from H. cinaedi specimens.  In brief, two volumes of RNA Protect Bacterial Reagent (Qiagen) were added to each culture.  Pelleted bacteria were re-treated (10 minutes, 65ºC) with Max Bacterial RNA Enhancement Reagent (Ambion), followed by TRIzol extraction and spin column purification (miRNeasy Mini Kit, Qiagen) of total RNA.  Nucleic acid quality was assessed by 260/280 absorbance and 23S/16S rRNA ratios. Illumina library preparation was performed at the Vanderbilt University sequencing core (VANTAGE – Vanderbilt Technologies for Advanced Genomics).  From each specimen, ribosomal RNA was depleted with the Gram-Negative Ribo-Zero rRNA Removal Kit (Epicenter). cDNA libraries  was constructed for Illumina sequencing through the TruSeq Stranded mRNA Library Prep Kit (Illumina).
Experimental Factor: Monophasic F12 medium
growth condition
Experimental Factor: H2-supplemented microaerobic
environmental stress