title: CFBP 1430 WT in vitro (M9 sucrose)
organism: Erwinia amylovora CFBP1430
description: Protocols: Growth at 28°C with continuous shaking to the mid-log phase in liquid minimal M9 medium (Difco) supplemented with sucrose (0.4 g l-1). RNA was extracted using the NucleoSpin RNA II kit (Macherey Nagel) following manufacturer's instructions. Total RNA samples were treated with DNase I (Thermo Scientific). Quality and concentration was determined using the Bioanalyzer 2100 (Agilent). Samples were pooled to the required amount for further processing. For the cDNA bacterial transcripts from the in planta RNA extract, first the polyA+ RNA was removed by oligo(dT) chromatography. The polyA- RNAs or RNA extracts from pure bacterial cultures were then treated with Terminator exonuclease (TEX) in order to enrich for bacterial transcripts carrying 5'-triphosphate. The terminator resistant transcripts were fragmented by ultrasound treatment (4 pulses of 30 s at 4°C) and then poly(A) tails were added to the 3' ends of the RNA fragments using poly(A) polymerase. The polyadenylated RNA fragments were further treated with RNA 5' polyphosphatase to remove 5'triphosphate groups from the 5' fragments After ligation of a RNA oligonucleotide to the 5' monophosphate of the RNA fragments, first-strand cDNA was synthesized using an oligo(dT)-linker primer and M-MLV H- reverse transcriptase. Finally, the cDNA was PCR amplified using a high fidelity DNA polymerase and 15 cycles. The cDNAs were purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and analyzed by capillary electrophoresis.