title: RNA-seq of E.coli: pspA-null mutant
design: The total RNA of wild type and pspA cells from middle stationary phase were extracted by RNAprep Pure Cell/Bacteria Kit (TIANGEN) according to the manufacturers instructions. The mRNA was purified from total RNA using probes to remove rRNA. Next, the mRNA was fragmented using divalent cations under elevated temperature in First Strand Synthesis Reaction Buffer(5X) to desired length reverse transcripted into first strand cDNA. The single strand cDNA was used for double strand DNA synthesis followed by end repair, dA-tailing, adaptor ligation and PCR amplification. In order to select cDNA fragments of preferentially 370~420 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA).