ena-STUDY-IATA-CSIC-06-08-2014-12:25:13:692-18
Transcriptome analysis of Bifidobacterium pseudocatenulatum CECT 7765 growing with Galacto-oligosaccharides (GOS) as carbon source.
Bifidobacterium pseudocatenulatum CECT 7765 was grown in glucose-free MRS media supplemented with 1% lactulose-derived Galacto-oligosaccharides (GOS) under anaerobic conditions at 37ºC. Control samples consisted in Bifidobacterium pseudocatenulatum CECT 7765 grown in MRS supplemented with 1% Glucose. Three independent replicates of cell cultures were collected in early log phase after approximately 8h growth. Cells were recovered from 15 mL media at OD600= 0.3-0.4 and stored on ice until RNA extraction was done. A commercial Gram-positive DNA extraction kit was used where lysis step were performed by using 500ug Lysozyme and 20U mutanolysin in addition to lysis buffer from manufacturer. RNA from replicates was mixed in equal molarity to produce one single mix sample from GOS treatment and another from Glucose treatment. Approximately 30 ug of total RNA were sent to Eurofins Genomics (Ebersberg, Germany) where samples were rRNA depleted using Ribo-Zero Magnetic Kit (Gram-positive bacteria). cDNA was synthesized from GOS- and Glucose-derived mRNA-enriched samples and they were sequenced in multiplex fashion in one MiSeq Illumina lane with 150bp configuration. Reads were mapped against the reference Bifidobacterium pseudocatenulatum CECT 7765 genome using blast-based strategies. Differential gene expression was inferred from z-score transformation of GOS/Glucose ratios for every single gene expressed. Functional annotation of genes was assisted by using KEGG annotation.
Bifidobacterium pseudocatenulatum CECT 7765 was grown in glucose-free MRS media supplemented with 1% lactulose-derived Galacto-oligosaccharides (GOS) under anaerobic conditions at 37ºC. Control samples consisted in Bifidobacterium pseudocatenulatum CECT 7765 grown in MRS supplemented with 1% Glucose. Three independent replicates of cell cultures were collected in early log phase after approximately 8h growth. Cells were recovered from 15 mL media at OD600= 0.3-0.4 and stored on ice until RNA extraction was done. A commercial Gram-positive DNA extraction kit was used where lysis step were performed by using 500ug Lysozyme and 20U mutanolysin in addition to lysis buffer from manufacturer. RNA from replicates was mixed in equal molarity to produce one single mix sample from GOS treatment and another from Glucose treatment. Approximately 30 ug of total RNA were sent to Eurofins Genomics (Ebersberg, Germany) where samples were rRNA depleted using Ribo-Zero Magnetic Kit (Gram-positive bacteria). cDNA was synthesized from GOS- and Glucose-derived mRNA-enriched samples and they were sequenced in multiplex fashion in one MiSeq Illumina lane with 150bp configuration. Reads were mapped against the reference Bifidobacterium pseudocatenulatum CECT 7765 genome using blast-based strategies. Differential gene expression was inferred from z-score transformation of GOS/Glucose ratios for every single gene expressed. Functional annotation of genes was assisted by using KEGG annotation.