Scriptseq complete kit bacteria

Approximately 3 × 10⁷ cfu of E. faecium E745 were inoculated into 14 ml of BHI broth and heat-inactivated serum, and grown at 37 °C until exponential phase. Cultures were centrifuged at room temperature (15 s; 21.380 g), and pellets were flash frozen in liquid N₂ prior to RNA extraction, which was performed as described previously [21 (link)]. The ScriptSeq Complete Kit (Bacteria) (Epicentre Biotechnologies, WI) was used for rRNA removal and strand-specific library construction. Briefly, rRNA was removed from 2.5 μg of total RNA. To generate strand specific RNA-seq data, approximately 100 ng of rRNA-depleted RNA was fragmented and reverse transcribed using random primers containing a 5′ tagging sequence, followed by 3′ end tagging with a terminal-tagging oligo to yield di-tagged, single-stranded cDNA. Following magnetic-bead based purification, the di-tagged cDNA was amplified by PCR (15 cycles) using ScriptSeq Index PCR Primers (Epicentre Biotechnologies, WI). Amplified RNA-seq libraries were purified using AMPure XP System (Beckman Coulter) and sequenced by a 100 bp paired end reads sequencing run using the Illumina HiSeq 2500 platform (University of Edinburgh, United Kingdom). Data analysis was performed using Rockhopper [64 (link)] using the default settings for strand specific analysis.

P. putida strains were precultured in MM containing 5 mM 3CBA until exponential growth phase (OD = ~0.6) and transferred into fresh medium with 100-fold dilution. The cells of four replicate cultures were collected either at exponential phase (OD = ~0.6) or from late-stationary phase cells grown for 96 h on 3CBA, and then further stimulated for 4.5 h with additional 5 mM 3CBA (REG phase). Collected cells were immediately treated with RNAlater (Ambion). Total RNA was extracted using the hot phenol method^{59 (link)}, and the residual co-extracted genomic DNA was digested by incubation with Turbo DNase (Life Technologies) following manufacturer’s instructions. The RNA was further purified using the RNeasy MiniElute cleanup kit (Qiagen), and rRNA was specifically depleted using Ribo-Zero rRNA removal kit Bacteria (Epicentre). The quality and quantity of RNA were measured using a Fragment Analyzer (Advanced Analytical) and a High Sensitivity RNA Analysis Kit (Advanced Analytical). cDNA libraries were generated using a ScriptSeq Complete Kit Bacteria (Epicentre) following strand-specific library preparation protocol. The indexed cDNA libraries were pooled and sequenced on Illumina HiSeq. 2000 platform with 101-nt single-end reads.