GSM7775308: 1_Lcr35 + STS; Lacticaseibacillus rhamnosus; RNA-Seq
Total RNA was extracted by the TRIzol method. Harvested cells were immediately submerged in TRIzol reagent (Ambion) on ice (1 mL of TRIzol per 50 cm2 of culture dish surface area). The cells were disrupted using a Fastprep apparatus twice for 45 sec at speed 6. The samples were subsequently centrifuged for 10 min at 14,000 rpm and purified in TRIzol/chloroform (5:1) and chloroform. For RNA precipitation, the samples were incubated in isopropanol at −20°C overnight and centrifuged for 30 min at 10,000 g. RNA samples were washed in ethanol and resuspended in 30 μL of DEPC water (Ambion). Residual DNA in the RNA samples was removed by DNase I treatment (Ambion). The concentration of the RNA samples was determined by Invitrogen Qubit Fluorometer (Thermo Fisher Scientific), and RNA qualities were determined by Agilent RNA pico chip (Agilent Technologies, Santa Clara, California, USA). Sequencing and analysis of the RNA data were performed by the MGX Genomics platform (Montpellier France). Ribosomal RNA was removed from each total RNA sample and libraries were produced using the Stranded mRNA Prep Ligation Kit, and sequenced using NovaSeq flow cell SP in single-read 100 pb (Illumina, San Diego, California, USA).