GSM3466607: tyrR-rep2; Enterobacter ludwigii; RNA-Seq
A total of 0.5 ml of cell culture were transferred to a 2 ml tube containing 1 ml RNAprotect Bacteria Reagent (Qiagen), incubated 5 minutes at room temperature, and then pelleted by centrifugation. Cell pellets were resuspended with 15 mg/ml lysozyme and 15 mg/ml Proteinase K in 200 µl TE buffer to lyse cells for 10 min at 4°C. Total RNA was extracted using the RNeasy Mini Kit (Qiagen). Contaminating genomic DNA was removed by treating 5 µg total RNA in a 50 µl reaction containing 5 units Amplification Grade DNase I (Invitrogen, CA, USA) and 5 µl 10x DNase I digestion buffer and incubating at 37°C for 60 minutes. The reaction was terminated with 1 mM EDTA and heating at 70°C for 10 minutes. The DNase-treated RNA was purified using the Qiagen MinElute Cleanup Kit (Qiagen, Hilden, Germany) and eluted into 30 µl RNase-free water. DNA removal was verified by PCR. The final quality and quantity of RNA samples were measured on an Agilent Bioanalyzer-RNA 6000 Pico chip (Agilent Technologies, CA, USA). Construction of RNA-Seq libraries and sequencing were performed by the Genome Quebec Innovation Centre (McGill, Montreal, Quebec). Ribosomal RNA was removed using the Ribo-Zero rRNA Removal Kit for Gram-Negative Bacteria (Illumina, CA, USA). The resulting enriched mRNA was used to construct a 100 bp paired-end stranded Illumina library using the TruSeq RNA Library Prep Kit v2 (Illumina). The libraries were individually barcoded and sequenced together on a single Illumina HiSeq 2000 lane.
GEO Accession
GSM3466607