GSM1846680: R_s_stationary_phase_TEX_neg; Rhodobacter sphaeroides 2.4.1; RNA-Seq
Total RNA was isolated using the hot phenol method followed by two chloroform-isoamyl alcohol treatments and precipitation with sodium acetate and ethanol. RNA was resolved in RNase free water (Roth) and concentrations were determined at a NanoDrop 1000 Spectrophotometer (Peqlab). The sample was treated with DNaseI (Invitrogen) to remove DNA. The libraries were generated by Vertis Biotechnologie AG (Munich, Germany). The samples were poly(A)-tailed by using poly(A) polymerase. The 5'-PPP were removed using tobacco acid pyrophosphatase (TAP) followed by the ligation of the RNA adapter to the 5'-monophosphate of the RNA. First-strand cDNA synthesis was performed with an oligo(dT)-adapter primer and the M-MLV reverse transcriptase. The resulting cDNA was PCR-amplified to reach a concentration of 20-30 ng/μl using a high fidelity DNA polymerase. The cDNA was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and was analyzed by capillary electrophoresis. The primers used for PCR amplification were designed for TruSeq sequencing according to the instructions of Illumina