Instrument: Illumina HiSeq 2500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: Total RNA of SE1457, △phoU1 and △phoU2 were isolated using the RNeasy Mini kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. Briefly, the bacterial cultures (6h) were centrifuged at 5,000 x g for 5 min, then the pellets were washed twice in 0.9% saline. The culture was homogenized using 0.1 mm Zirconia-silica beads in a Mini-Bead beater (Biospec, Bartlesville, OK, USA) at 4800 rpm for 40 sec at 1 min intervals on ice for 5 times. The RNA extracted using the silica-based filter was purified by phenol-chloroform-isoamyl alcohol and precipitated with absolute ethanol. RNA samples of SE1457, △phoU1 and △phoU2 were treated with RNase-free DNase I (Takara) to prevent contamination with genomic DNA. The RNA quality was evaluated with the BioAnalyzer 2100 system (Agilent technologies, Deutschland GmbH). Ribosomal RNA was removed with the RiboZero rRNA removal kit, which is for gram-positive organisms prior to sequencing analysis. After depletion of the rRNA, fragmented RNA was used as a template for PCR with random primers. The cDNA libraries were prepared by using the mRNA-seq Sample Prep kit (Illumina). The concentration of the cDNA was measured by Qubit 2.0 Fluorometer, the fragment size (200–300 bp) verified on a BioAnalyzer 2100 system, and amplified using an Illumina cBot and sequenced on an Illumina HiSeq 2500 for 51 cycles according to manufacturer protocols.