BW25113 dcspCdcspE(kan)+pT7-5 extract a
Cell pellets were suspended in extraction buffer and cell extracts were prepared by sonication in the presence of pefabloc, RQ1 DNase, and RNasIn®plus. The cell extracts were samlped for total extract RNA-seq, and the rest were used for cell fractionation. RNA was extracted from the extracts and fractions and analyzed by RNA-seq. Libraries were prepared according to the Illumina protocol. Essentially, triplicate samples of rDNaseI treated RNA (1 µg) were fragmented using RNA fragmentation kit (Ambion, AM8740). Fragmented RNA was purified using AMPure magnetic beads (Agencourt A63881) at a 2.2/1 ratio and reverse transcribed at a final volume of 12 µL. Second strand cDNA synthesis was performed by addition of the following: 3 μL NEB2 10X buffer, 1.2 μL dNTPs 10 mM, 1.2 μL dATP 10 mM, 0.8 µL E. coli DNA polymerase (NEB, M0209), 1.6 μL RNaseH (NEB, M0297), 0.4 μL T4 DNA ligase (NEB, M0202). Final volume was adjusted to 30 μL using nuclease free water and the reaction was incubated at 16 °C for 2.5 h. Double strand cDNA was purified using magnetic beads, and undergone A-addition using KLENOW exo- (NEB, M0212) in NEB buffer 2 supplemented by 167 µM dATP for 30 minutes at 37 °C. Reaction product was purified using magnetic beads and ligated to adapters carrying the Illumina sequences using Quick ligation kit (NEB, M2200) as described. The resulting libraries were amplified with 14 cycles of PCR using the PFUultraII fusion (Agilent).