Illumina Genome Analyzer IIx sequencing; Transcriptome analysis of Vibrio vulnificus cells that are either exposed to HT29-methotrexate intestinal epithelial cells or grown in basal medium eagle
Transcriptome analysis of Vibrio vulnificus cells that are either exposed to HT29-methotrexate intestinal epithelial cells or grown in basal medium eagle
V. vulnificus MO6-24/O cells (approximately 2 X 10^8 colony forming unit) grown in basal medium eagle for 2 h were fixed with 2:1 volumes of RNAprotect Bacteria Reagent (Qiagen) and harvested. Total RNAs were isolated from the V. vulnificus cells using miRNeasy Mini Kit (Qiagen). Genomic DNA in the purified RNA was removed with digestion using TURBO^TM DNase (Ambion) to below PCR-detectable levels. RNA quality was determined using Bioanalyser (Agilent) 23S and 16S rRNA of total RNAs were depleted using MICROBExpress kit (Ambion). The enriched mRNAs were fragmented using Fragment, Prime, Finish Mix (Illumina). The mixtures were reacted at 94°C for 8 min and then incubated at 4°C.  Synthesis of first strand cDNA was prepared by mixing 0.5 μg of the enriched mRNA, First Strand Synthesis Act D Mix (Illumina), and Superscript® II polymerase (Invitrogen). The mixture was incubated at 25°C for 10 min and at 42°C for 45 min and the polymerase was finally inactivated at 75°C for 15 min. Remaining dNTPs were removed AMPure XP beads. Then, the second strand of cDNA was further synthesized using Second Strand Marking Master (Illumina). The mixture was incubated at 16°C for 60 min. Remaining dUNTPs were removed AMPure XP beads.  A single 3’ adenosine moiety was added to the cDNA fragments using A-Tailing mix (Illumina) The Illumina adapters (containing primer sites for sequencing and flowcell surface annealing) were ligated onto the repaired ends on the cDNA fragments using Ligation Mix (Illumina). The mixtures were incubated at °C for 10 min and then add the Stop Ligation Buffer (Illumina) for inactivation of ligation. Final mixtures were purified using AMPure XP beads.
Experimental Factor: basal medium eagle for 2 h (as negative control)
growth condition