Illumina HiSeq 4000 sequencing; RNAseq of Faecalibacterium prausnitzii A2-165 stimulated with cell-free supernatants from lactic acid bacteria.
RNAseq of Faecalibacterium prausnitzii A2-165 stimulated with cell-free supernatants from lactic acid bacteria.
We collected 15 mL of F. prausnitzii culture at the end of the exponential growth phase (T22), for each condition and replicate, and added it to 30 mL of RNA Protect bacterial reagent (Qiagen, Germany) at pH 6. This mixture was immediately vortexed and incubated for 5 minutes at room temperature. It was then centrifuged at 5000 x g for 10 minutes and the pellet was recovered and stored at -80°C until use. RNA was isolated by enzymatic and mechanical lysis followed by use of the RNAeasy Mini Kit (Qiagen, Germany). The RNA was concentrated during DNAse treatment with the RNeasy MiniElute Cleanup kit (Qiagen, Germany). Quantity and quality of the RNA was assessed with a NanoDrop® photometer (Thermo Scientific, USA) and an Agilent 2100 Bioanalyzer (Agilent Technologies, USA) respectively.  We used 1 µg of DNA-free RNA with a RIN value > 8.0 from each sample for ribosomal RNA removal and library construction with Ribo-zero and the Scriptseq v2 RNAseq Library preparation kit (Illumina, USA).  Quality control was performed on libraries with the Agilent High-Sensitivity DNA Kit (Agilent Technologies, USA).