GSM5322273: JFP169-T-20-Rep-1; Streptococcus sanguinis; RNA-Seq
For each RNA sample, 2 mL of fermentor culture was added to 4 mL RNAprotect Bacteria Reagent (Qiagen) and immediately vortexed for 10 sec. The sample was then incubated at room temperature for 5-90 min. The samples were then centrifuged for 10 min at 3,740 x g at 4°C. The supernatant was discarded and the samples stored at -80°C. RNA isolation and on-column DNase treatment were completed using the RNeasy Mini Kit and RNase-Free DNase Kit, respectively (Qiagen). RNA was eluted in 50 µL RNase-Free water (Qiagen). A second DNase treatment was then performed on the samples (Invitrogen). Total RNA quantity and integrity were determined using an Agilent Bioanalyzer RNA Pico assay.  All samples passed quality control assessment with RNA Integrity Numbers (RIN) above 8.  Two sequential rounds of ribosomal reduction were then performed on all samples using RiboMinus™ Transcriptome Isolation Kit (ThermoFisher).  The resulting depleted RNA was assessed using an Agilent Bioanalyzer RNA Pico assay to confirm efficient rRNA removal.  Stranded RNA-seq library construction was then performed on the rRNA-depleted RNA using the Kapa RNA HyperPlus kit for Illumina (Roche) following manufacturer's specifications for library construction and multiplexing. Final Illumina libraries were assessed for quality using an Agilent Bioanalyzer DNA High Sensitivity Assay and qPCR quantification was performed using Kapa Library Quantification kit for Illumina (Roche).