GSM4183209: GAM1GAM2.1.3; Ruminococcus gauvreauii; Ruminococcaceae bacterium IPLA60002; RNA-Seq
Total RNA was extracted from the pellets that were resuspended in 200 μl of lysozyme (concentración) and mutanolysin (concentración) solution and incubated at 37°C for 1 h. Then, 800 μl of QIAzol (Qiagen) were added and placed in a tube containing 0.8 g of glass beads (106 μm diameter; Sigma). The cells were lysed by shaking the mix on a BioSpec homogenizer (BioSpec Products, Inc., USA) for 2 min followed by 2 min in ice. This step was repeated three times. Samples were further treated with chloroform and then centrifuged at 12,000 rpm for 15 min at 4°C. The upper phase containing the RNA was recovered. Samples were further purified using the RNeasy Mini Kit (Qiagen) following the manufacturer's protocol. Quality and integrity of the RNA was checked by an Agilent 2200 Tape Station Nucleic Acid System (Agilent Technologies, Palo Alto, Calif., USA). The amount of RNA was quantified in a Qubit™ 4 fluorometer with Qubit RNA assay kits (Thermo Fisher Scientific, Inc., Waltham, MA, USA). One to 2.5 μg of total RNA was depleted using Ribo-Zero rRNA removal kit (Illumina) to remove rRNA according to the supplier's instructions. The yield of rRNA depletion was checked in the Agilent Tape Station and rRNA-depleted RNA samples were treated with the TruSeq Stranded mRNA sample preparation kit (Illumina). Size evaluation and quantification of RNA samples was performed as above described. Whole transcriptome library was sequenced by means of a NextSeq 550 Sequencing System (Illumina) using a High Output kit v2.5 (75 cycles) flow cell