title: GSM3657725: HOCl_0min_repB; Lactobacillus reuteri; RNA-Seq
library_construction_protocol: Cells (7 ml) were harvested at 0 min (no addition of stress; the tube which did not receive any oxidant), 5 min, 15 min and 30 min after addition of oxidants by mixing with 7 ml ice-cold isopropanol anaerobically to stop transcription, and then removing the samples from the anaerobic chamber and centrifuging at 1500 x g for 15 minutes at 4°C. The supernatant was discarded and the pellet was resuspended in the remaining isopropanol and transferred to 1.5 ml microfuge tubes. The cells were then centrifuged at 4000 x g for 10 minutes at 4°C. The supernatant was removed completely and the samples were stored at -80°C for further processing. Total RNA was extracted using the RiboPure™ RNA Purification Kit for bacteria (Ambion) following the manufacturer's protocol. All RNA samples were DNAse treated with the optional DNAse treatment kit provided with the Ambion RiboPure Bacteria kit to remove any contaminating genomic DNA. RNA-sequencing was performed on the Illumina HiSeq2500 using the Rapid Run v2 sequencing chemistry and flow cells as described by the manufacturer (Illumina Inc., San Diego, CA).  Briefly, the quality of the total RNA was assessed using the Agilent 2100 Bioanalyzer.  RNA with a RNA Integrity Number (RIN) of 7.0 or above was used for sequencing library preparation. We used the Agilent SureSelect Strand Specific mRNA library kit as per the manufacturer's instructions (Agilent, Santa Clara, CA).  Cluster generation was performed according to the manufacturers recommendations for onboard clustering (Illumina).  Paired end 50 bp sequencing runs were used to allow for better alignment of the sequences to the reference genome.