Bacterial RNA-seq sample preparation and sequencing.

RNA-seq was carried out using a single sample from each group to generate profiling information that could be validated and confirmed by using qPCR and metabolomics analyses. Total bacterial RNA from feces samples was isolated with ZR Soil/Fecal RNA MicroPrep (Zymo research) following the manufacturer’s instructions. Briefly, the sample was suspended in RNA lysis buffer and lysed by the mixer (Retsch). The supernatant was transferred to a RNA-binding column and washed several times with RNA wash buffer. In-column DNaseI digestion at 25°C for 15 min was performed to eliminate DNA contamination in the sample. The presence of genomic DNA contamination was assessed by PCR with universal 16S rRNA gene primers. Before RNA-seq library preparation, rRNA was removed from 2 μg of total bacterial RNA with Ribo-Zero Bacteria kit (Illumina). 100 ng of purified RNA was used for RNA-seq library preparation as described previously⁶⁰. The cDNA was synthesized by reverse transcription with SuperScript III (Life technologies) and second-strand synthesis (New England Biolabs). The sequencing library was generated from purified cDNA with Nextera XT DNA library preparation kit (Illumina) and amplified by PCR. Cycling conditions were 72°C for 3 min, 95°C for 30 s, followed by 16 cycles of 95°C for 10 s, 55°C for 30 s, and 72°C for 30 s. A condition of 72°C for 5 min was used for the final elongation step. Libraries with different indexes were pooled and sequenced on an Illumina NextSeq at the Scripps Research Institute next generation sequencing core. 4,453,883, 8,956,883, and 9,166,765 reads were generated from the CHD, WD, and WD + c[wLwReQeR] samples, respectively. Of these reads, 1,367,533 were mappable for CHD (30.7% of total generated reads), 4,411,184, were mappable for WD (49.2% of total generated reads), and 4,470,591 were mappable WD + c[wLwReQeR] (48.8% of total generated reads).