Powermicrobiome rna isolation kit

At each sample point, growth in cultures was stopped by adding 10% phenol:chloroform (Fisher Scientific) in ethanol solution pre-chilled to -80°C in a 1:10 volume to the sample. The tubes were vortexed briefly and then centrifuged immediately for 10 min at 4,696×g and 0°C. The supernatants were removed and the resulting pellets were stored at -80°C for less than 2 weeks. Total RNA was isolated and purified using the PowerMicrobiome^™ RNA Isolation kit (MO BIO Laboratories, CA, USA) per the manufacturer’s protocol. RNA integrity numbers (RINs) were determined using the 2100 Bioanalyzer (Agilent, CA, USA). Samples with a RIN between 9.7 and 10 and an RNA concentration >100 ng/μl were sent to Genome Québec (Montréal, QC, Canada) for rRNA-depleted Illumina TruSeq RNA library prep and TruSeq stranded total RNA 100 bp paired-end sequencing on the Illumina HiSeq 2000 platform.

Microbial DNA was extracted from frozen fecal samples using the PowerMicrobiome RNA isolation kit (Mo Bio Laboratories, Inc., Carlsbad, CA) as described previously (24 (link)). 16S rRNA genes were amplified using the 515F/806R primer set, targeting the V4 hypervariable region (29 (link)). Sequencing was performed using the Illumina MiSeq platform with sequencing kit MiSeq v2, producing 250-bp paired-end reads. For sequence analysis, fastq sequences were merged using FLASH version 1.2.10 (30 (link)) and quality filtered (threshold: >90% of nucleotides should have a quality score of ≥25) with the FASTX-Toolkit v0.0.14 (http://hannonlab.cshl.edu/fastx_toolkit/). Chimera removal was performed using the UCHIME algorithm in USEARCH v6.0.307 (31 (link)), and taxonomical assignment of sequences was performed with the RDP classifier v2.12 (32 (link)). Phylum-to-genus matrices were subsequently created using Perl scripts. Samples were rarefied to 10,000 randomly selected reads, with samples with <10,000 reads (n = 8) excluded from the analysis.

Fecal samples were collected from the resected colon and stored at −80 °C within 2 h after sampling. The total amount of fecal content with a median (interquartile) weight of 88 (10–137.5) mg was used for bacterial DNA extraction. The PowerMicrobiome RNA Isolation Kit (Mo Bio Laboratories, Carlsbad, CA, USA) was used with an adapted protocol. Briefly, samples were homogenized with the use of mechanical beating in a lysis buffer containing 1% β-mercaptoethanol. In addition, the samples were incubated at 90 °C for 10 min. The supernatants containing DNA and RNA from both bacterial and murine cells were then bound to a spin filter membrane with the use of centrifugation. After three washing steps, the nucleic acids were eluted from the membrane in 100 µl RNase-free water and were stored at −80 °C. Bacterial DNA was quantified with a Qubit™ 2.0 fluorometer (Life Technologies, Grand Island, New York). After PCR amplification, Fragment analyzer™ (Advanced Analytical Technologies, Ames, Iowa) was used for quality control and quantification of the libraries.

The liquid samples were acquired from the triplicate reactors before, 18 hours after and 36 days after H₂ addition (Sample point 1, 2, and 3, respectively). For all the samples, the genomic DNA was extracted with PowerSoil® DNA Isolation Kit and the total RNA was extracted PowerMicrobiome® RNA Isolation Kit (Mo Bio Laboratories, Inc., Carlsbad, USA). All the extractions were performed with additional phenol cleaning steps in order to improve the quality of the extractives. The ribosome RNA was removed from total RNA samples with Ribo-Zero® rRNA Removal Kit (Bacteria) (Illumina, San Diego, USA). The DNA and RNA samples were sent to Ramaciotti Centre for Genomics (UNSW, Sydney, Australia) for cDNA construction, library preparation, and sequencing (Illumina NextSeq).

Bacterial pellets were lysed with 1 mg/ml lysozyme (Sigma-Aldrich, Isle d’Abeau Chesnes, France) for 5 min at 25 °C, followed by Total RNA extraction using the RNeasy Mini Kit (Qiagen, Courtaboeuf, France) according to the manufacturer’s recommendations. RNA concentration and purity was evaluated by measuring the absorbance ratio at 260/280 nm and 260/230 nm using a Nanodrop spectrophotometer (Labtech, Palaiseau, France). The Ratio Integrity Number (RIN) was evaluated using 2100 Bioanalyzer® (Agilent Technologies, Massy, France) and only samples with a RIN greater than 8 were hybridized on the microarray.
Total RNA of rumen derived consortium was extracted in two steps from nitrogen frozen samples using the PowerMicrobiome RNA isolation kit (MoBio Laboratories, Carlsbad, CA, USA) [36 (link)]. RNA purification was performed using AllPrep DNA/RNA minikit (Qiagen), according to the manufacturer’s recommendations.

The extraction of total RNA from feces was based on the method described by Reck et al.^{51 (link)}, and we used commercially available kits to isolate total RNA from small amounts of stool samples (PowerMicrobiome™ RNA Isolation Kit, MoBio Laboratories, Germany). In short, approximately 0.25 g of feces was transferred to a glass bead tube (0.1 mm). Shortly before the feces sample was completely thawed on ice, added β-mercaptoethanol to the MoBio lysis buffer to make the final concentration of 10 μl/ml in the tube, and homogenized on the Vortex-Genie 2 Mixer at the highest speed for 10 min. The sample was centrifuge at 13,000×g for 1 min at room temperature, and further steps included DNAse I treatment of the samples and elution of RNA in nuclease-free water, which were performed according to the manufacturer's instructions. The RNA concentration was measured using Nanodrop ND 2000 (Thermo Scientific, USA).
The miRNA profiles in human feces were determined by NanoString nCounter System (NanoString Technologies) and the nCounter Human-v2 miRNA Panel with ~ 800 unique miRNA barcodes. Assays and spike-in controls were used for normalization based on identical amounts of input RNA.

RNA from bioreactor-derived bacteria was isolated using PowerMicroBiome RNA isolation kit from MO Bio Laboratories (San Diego, CA). Briefly, the bacteria pellets were combined with lysis buffer and glass beads. Subsequently they were lysed for 5 minutes in Qiagen TissueLyser II (Valencia, CA) at 30 Hz. Further, the process included inhibitor removal step and standard on-column purification was carried out according to manufacturer’s instructions. RNA purification included on-column DNAse treatment for 15 minutes at room temperature. Subsequently RNA concentration and quality were determined by RNA electrophoresis on Agilent bioanalyzer (Santa Clara, CA).