RNA-Seq of Pseudomonas: PAO1 deleted for the 9bp3 in PA5438
1 g total RNA was used for following library preparation. Remove rRNA, the mRNA fragmentation was performed using divalent cations and high temperature. Priming was performed using Random Primers. First strand cDNA and the second-strand cDNA were synthesized. The purified double-stranded cDNA was then treated to repair both ends and add a dA tailing in one reaction, followed by a TA ligation to add adaptors to both ends. Size selection of Adaptor-ligated DNA was then performed using DNA Clean Beads. Then libraries with different indexs were multiplexed and loaded on an Illumina HiSeq 2000 instrument for sequencing using a paired-end configuration according to manufacturers instructions.