Bacteria were lysed by constantly grinding in the presence of liquid nitrogen and total RNA was extracted using TRIzol reagent (Invitrogen). RNA was fully treated with RNase-free DNase I (Takara) to prevent contamination of trace genomic DNA [8 (link), 42 (link)]. The RNA quality was evaluated by the BioAnalyzer 2100 system (Agilent) and ribosomal RNAs were removed with the RiboZero rRNA removal kit (Epicenter) for gram-positive organisms prior to sequencing analysis. After deprivation of the rRNA, RNA was fragmented and used as a template for a randomly primed PCR.
Strand-specific cDNA libraries were prepared by standard techniques for subsequent Illumina sequencing using the mRNA-seq Sample Prep kit (Illumina). The concentration of the cDNA library was detected by Qubit®2.0 Fluorometer and verified for appropriate fragment size (200–300 bp) on a BioAnalyzer 2100 system (Agilent). Samples were amplified onto flowcells using an Illumina cBot and sequenced on an Illumina HiSeq 2500 for 51 cycles according to manufacturer protocols. Raw sequencing data was processed using the data collection software provided by Illumina [43 (link)].
Strand-specific cDNA libraries were prepared by standard techniques for subsequent Illumina sequencing using the mRNA-seq Sample Prep kit (Illumina). The concentration of the cDNA library was detected by Qubit®2.0 Fluorometer and verified for appropriate fragment size (200–300 bp) on a BioAnalyzer 2100 system (Agilent). Samples were amplified onto flowcells using an Illumina cBot and sequenced on an Illumina HiSeq 2500 for 51 cycles according to manufacturer protocols. Raw sequencing data was processed using the data collection software provided by Illumina [43 (link)].
Free full text: Click here
