GSM7729829: BACI358, Resistant, Time 60 minutes, Replicate B; Bacillus anthracis; RNA-Seq
2 volumes of RNAprotect Bacteria Reagent was added to each tube and incubated at room temperature for 5 minutes at indicated time points. For all reactions tubes were centrifuged at 5000 x g for 10 minutes. The supernatant was decanted, and pellets were extracted or stored at -80°C until processing. Pellets were resuspended in 100µl 1xTE containing 15 mg/ml lysozyme and 10 µl Proteinase K. Suspended pellets were incubated at room temperature for 10 minutes. Then, 700 µl of buffer RLT was added to suspended pellets along with approximately 100 µl of 0.5 mM glass beads and cells were incubated at 95°C for 5 minutes, bead beaten for 10 minutes, then centrifuged at max speed for 30 seconds. 760 µl of supernatant was added to new 2 ml Eppendorf tubes containing 590 µl of 80% ethanol. Samples were then extracted utilizing the RNeasy kit protocol and an on-column DNase treatment according to manufacturer's protocols Extracted RNA was quantified using the Qubit 2.0 Fluorometer and Qubit RNA HS assay. RNA was spiked with the ERCC control, an external RNA control, and input into the QIAseq FastSelect – 5s/16s/23s kit to remove bacterial ribosomal RNA prior to sequencing. Sequencing libraries were prepared using the QIAseq Stranded total RNA library kit according to manufacturer's instructions. Libraries were quantified using the High Sensitivity D1000 kit on the 4200 Tapestation System. Four barcoded libraries were combined and diluted to a 4 nM library pool, denatured with NaOH, and further diluted to a final library concentration of 12 pM prior to sequencing. Pooled libraries were sequenced using the MiSeq Instrument and MiSeq reagent kit version 3–150 cycles.