Rg-Glc-D
Mediterraneibacter gnavus ATCC 29149
Protocols: The RNA was stabilized prior to extraction by adding 1/5 vol of phenol (pH 4.3): ethanol (1:9) mixture to 1 vol of bacterial culture then incubating 30 min on ice and finally pelleting the cells for 5 min at 10,000 g at 4ºC. Cell pellets were stored at -80ºC before extraction. Cultures of R. gnavus ATCC 29149 and/or R. bromii L2-63 were performed, at 37C in anaerobic conditions, in a minimum medium called YCFA supplemented with a single carbon source (Glucose, soluble starch or resistant starch). At mid- to late exponential phase, 5mL of culture was collected for total RNA extraction. The RNA extraction was performed by a method using phenol and chloroform and adapted from Sambrook and colleagues (Sambrook et al., 1989). Genomic DNA contamination was removed by DNAse treatment using TURBO DNA-free kit (Life Technologies Ltd, Paisley, UK) according to supplier's recommendations. The purity, quantity and integrity of the DNase-treated RNA were assessed with NanoDrop 2000 Spectrophotometer and with Agilent RNA 600 Nano kit on Agilent 2100 Bioanalyzer (Agilent Technologies, Stockport, UK). Ribodepletion was then carried out using Ribo-Zero® rRNA Removal kit for bacteria according to supplier's advice (Illumina, Cambridge, UK); efficiency assessment of the ribodepletion was performed by quantifying RNA before and after rRNA removal using the Qubit RNA HS assay on Qubit® 2.0 fluorometer.The rRNA removal was confirmed with a Nano chip run on a Bioanalyzer 2100 (Agilent). The resulting ribosomal depleted RNA was then fragmented for 8 min at 94ºC using the Elute, Fragment, Prime buffer from Illumina TruSeq RNA kit. These conditions produced final libraries of around 370 bp. The samples were then processed following the standard TruSeq RNA protocol. The 15 Illumina libraries were normalized and equimolar pooled to 11 nM using elution buffer (Qiagen).The library pool was then diluted to 2 nM with NaOH and 5 μL transferred into 995 μL HT1 (Illumina) to give a final concentration of 10 pM. A portion (120 μL) of the diluted library pool was then transferred into a 200 μL-strip tube, spiked with 1% PhiX Control v3 and placed on ice before loading onto the Illumina cBot.