Illumina HiSeq 2500 paired end sequencing; RNAseq reveals cooperative metabolic interactions from co-culture of acetogenic and amylolytic human colonic bacteria.
RNAseq reveals cooperative metabolic interactions from co-culture of acetogenic and amylolytic human colonic bacteria.
Co-cultures of R. bromii with B. hydrogenotrophica and the respective mono-cultures in a continuous flow anaerobic fermentor system.  Total RNA was extracted  using phenol-chloroform extraction followed by ethanol precipitation. DNA contamination was removed using DNAse-treatment with TURBO DNA-free kit (Invitrogen). RNA concentration and purity were determined spectrophotometrically using Nano drop Spectrophotometer ND-1000, BioAnalyzer 2100  and QUBIT. Ribosomal RNA was removed from the samples using the Ribo-Zero rRNA removal kit for bacteria (Illumina) according to manufacturer's protocol. mRNA was then purified using RNeasy MinElute kit (Qiagen). A total of nine RNA libraries (three replicates each of R. bromii monoculture, B. hydrogenotrophica monoculture and R. bromii - B .hydrogenotrophica co-culture) were prepared using the adapted TruSeq RNA protocol (Illumina 15026495 Rev.B). The library preparation involved QC of the RNA using Bioanalyzer 2100 (Agilent Technologies Inc.) with the Pico kit (Agilent) on the mRNA setting to detect any potential rRNA contamination. The ribo-depleted RNA was chemically fragmented and double strand (ds) cDNA was synthesized using random hexamers in the presence of reverse transcriptase enzyme. The samples were then end repaired, tailed, adaptor-ligated, fractionated, purified and enriched. The insert size of the libraries was verified by running an aliquot of the DNA library on a PerkinElmer GX using the High Sensitivity DNA chip (PerkinElmer CLS760672) and the concentration was determined by using a High Sensitivity Qubit assay and q-PCR.
Experimental Factor: growth condition
monoculture