Following centrifugation of the lysate, nucleic acids were recovered from the aqueous phase and re-extracted with chloroform. DNA was selectively digested and the RNA was purified by using the RNeasy® mini kit (Qiagen) as described in the manufacturer instructions. A detailed protocol is provided in the supplementary information (See Additional file 3: Supplementary Methods). An equivalent of 1 mg of each fecal sample was used for RNA quantification

using a NanoDrop ND-1000 Spectrophotometer (Nucliber). The RNA was then examined by microcapillary electrophoresis using an Agilent 2100 Bioanalyzer with the RNA 6000 Nano Kit. The RNA quality was determined by the RNA integrity number (RIN), which is calculated from the relative height and area of the

16S and 23S RNA peaks and follows a numbering system from 1 to 10, being 1 the most degraded profile and 10 the most intact [14, 19]. Assessing the this website quantity and quality of genomic DNA Aliquots (250 mg) of each fecal sample were suspended in 0.1 M Tris (pH 7.5), 250 μl of 4 M guanidine thiocyanate and 40 μl of 10% N-lauroyl sarcosine. DNA extraction was conducted by mechanical MK-4827 cost disruption of the microbial cells with glass beads and recovery of nucleic acids from clear lysates by alcohol precipitation, as previously described in Godon et al. [20]. An equivalent of 1 mg of each fecal sample was used for DNA quantification using a NanoDrop ND-1000 Spectrophotometer (Nucliber). DNA integrity was examined by microcapillary electrophoresis using an Agilent 2100 Bioanalyzer with the DNA 12,000 kit, which resolves the distribution of double-stranded DNA fragments up to 17,000 bp in length. Assessment of microbial composition through 16 S rRNA gene survey In order to analyze bacterial composition, the V4 hypervariable region of the 16 S rRNA gene was amplified from the genomic DNA extracted from these fecal samples by using two universal primers: V4F_517_17 (5’-GCCAGCAGCCGCGGTAA-3’) [21] and V4R_805_19 (5’-GACTACCAGGGTATCTAAT-3’) [22]. Multiplex identifiers (MIDs), which were used to perform

tag pyrosequencing, were included upstream the forward primer sequence (V4F_517_17). PCR amplification was run in a Mastercycler gradient (Eppendorf) at 94°C for 2 min, followed by 35 cycles of 94°C for 30 sec, 56°C for 20 sec, 72°C for 40 sec, and a final cycle of 72°C for 7 min. PCR products were purified using PCR Purification kit (Qiagen, Spain) and subsequently sequenced on a 454 Life Sciences (Roche) Genome Sequencer FLX platform (UCTS, Hospital Vall d’Hebron, Barcelona, Spain). Sequence analyses were performed using the Qiime pipeline [23]. Sequences were deposited in Genbank (Genbank: BIBW2992 SRA055900). Uclust [24] was used to cluster sequences into OTUs (Operational Taxonomic Unit, taxa or species) at 97% sequence identity.