Ileostomy effluent samples collected for this analysis (see above) were thawed and homogenised while remaining cooled on ice using Ultra-Turrax t50 (IKA, Germany). Ten millilitres of the homogenised samples was transferred to a tube and larger debris was removed by low-speed centrifugation (3’, 500×g, 4 °C, with the centrifuge’s breaks switched off). Subsequently, bacteria and smaller-sized debris were pelleted by higher speed centrifugation (10’, 8000×g, 4 °C) and the pellet obtained was immediately resuspended in 100 μl of phenol–chloroform–isoamyl alcohol (pH 6.5–8.0, Sigma, Germany) followed by RNA extraction using the RNeasy PowerMicrobiome Kit (Qiagen, Germany) according to the manufacturer’s instructions. Extracted RNA was stored at – 80 °C. RNA quality and quantity were analysed by agarose gel electrophoresis as well as using the TapeStation 2200 (Agilent Technologies, CA, USA). Obtaining RNA of sufficient quality in these samples was challenging, resulting in a restricted and unbalanced sample size for this analysis. Most of the successfully sequenced samples were taken at the start or end of the fermented product intervention periods and only for three subjects a complete sample set (all 6 samples) could be analysed. The 250~300 bp insert cDNA library with rRNA removal (Ribo-ZeroTM Magnetic Kit, Illumina, USA) and sequencing were performed by Novogene (Hong Kong) using the HiSeq2500 platform (PE150, 12 G raw data/sample). HUMAnN2, with the default settings [35 (link)], was used for the functional profiling of the metatranscriptome datasets by mapping against UniRef90 protein database (updated global profiling of the Human Microbiome Project [36 (link)]) and MetaCyc database 19.1 [37 (link)] to obtain the bacterial pathway abundances (Functional Metatranscriptome Mapping) combined with taxonomic profiling by the included taxonomic identification tool MetaPhlAn2.
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