Epicentre scriptseq v 2 rna seq library preparation kit

RNA was isolated as previously described (83 (link)). Briefly, RNA was extracted from frozen cell pellets using a Direct-zol RNA Miniprep kit (Zymo Research) with the addition of an extra step for cell disruption using glass beads. The quality and concentration of the isolated RNA were verified both by gel electrophoresis and by using a NanoDrop spectrophotometer (Thermo Scientific). Due to the inability to isolate high-quality RNA from any of the late stationary planktonic samples, this time point was not included in the transcriptomic analysis. Genomic DNA was removed from the remaining total RNA samples using a Turbo DNA-free kit (Ambion). rRNA was removed from the remaining sample using a Ribo-Zero Gram-positive Bacteria rRNA removal kit (Epicentre Technologies) and purified with an Agencourt RNAClean XP kit (Beckman Coulter, Inc.). cDNA libraries were prepared from the purified RNA using a Epicentre ScriptSeq v 2 RNA-seq library preparation kit (Epicentre Technologies). The resulting cDNA was purified using an Agencourt AMPure XP system (Beckman Coulter, Inc.), and then quality and quantity were verified using an Agilent 2100 Bioanalyzer (Agilent Technologies).

Depleted RNA was used for RNA-Seq library preparation with the Epicentre ScriptSeq v2 RNA-Seq Library Preparation Kit (Epicentre-Illumina, CA) following the manufacturer’s protocol (EPILIT329 Rev. C). Agencount AMPure beads (Beckman Coulter, IN) were used to purify the cDNA, and unique indexes were added during 13 cycles of library amplification. The final RNA-Seq libraries were purified with Agencount AMPure beads (Beckman Coulter, IN) and quantified with a Qubit fluorometer (Life Technologies, CA). The library quality was assessed on a Bioanalyzer DNA 7500 DNA Chip (Agilent, CA), and samples were pooled and diluted. Two paired end sequencing runs were completed on an Illumina MiSeq Instrument (Illumina, CA) using the standard protocol. Raw sequencing fragment reads were mapped in CLC Genomics Workbench 9.0.1 (CLC bio, Denmark) using default prokaryote settings to genome [GenBank:CP000568.1] and the resulting unique gene read counts output was used for differential expression analysis with the R package DeSeq2 according to published methodology23 (link). RNA-Seq gene expression as well as raw sequencing and mapped reads data have been deposited in NCBI GEO accession number GSE87407.