title: RNA-Seq of Solanum tuberosum infected with Pectobacterium atrosepticum, stem cross section, replicate 3
design: Sterile virus-free potato plants were axenically grown on Murashige and Skoog medium in tubes in a growth chamber with a 16-h light/8-h dark cycle photoperiod. Apical stem parts were cut and rooted on sterile vermiculite for 14 days and then planted into the soil in 300 ml pots. Four weeks after planting into the soil, plants were infected with Pectobacterium atrosepticum SCRI1043 (Pba) and transferred to glass chambers to provide high humidity. Pba was cultured in Luria-Bertani (LB) broth on a rotary shaker (180 rpm) at 28C until the early stationary phase (~210^9 colony-forming units (CFU/ml)). For plant inoculation, bacterial cells were washed twice with and suspended in sterile 10 mM MgSO4 up to a density of ~210^7 CFU/ml. Ten l of the obtained bacterial suspension or sterile 10 mM MgSO4 were injected into the middle parts of plant stems using syringe. Three days after plant inoculation, the samples were collected for analyzes. For the in-fected plants, the asymptomatic zone located 1 cm below the necrotic zone were collected. Plant material was ground in liquid nitrogen. The obtained powder was resuspended in 1 mL of ExtractRNA Reagent (Evrogen, Russia) and the subsequent procedures were performed according to the manufacturers instructions. Residual DNA was eliminated using a DNA-free kit (Life Technologies, Carlsbad, USA). RNA quantity and quality were analyzed using a Qubit fluorimeter (Life Technologies, Carlsbad, USA) and Qsep100 DNA Analyzer (Bioptic, Taiwan), respectively. For mRNA enrichment, total RNA (3 g) was processed using Ribo-Zero rRNA Removal Kit (Plant) (Illumina, USA) and then Ribo-Zero rRNA Removal Kit (Gram-Negative Bacteria) (Illumina, USA). cDNA libraries were prepared using a NEB-Next Ultra II Directional RNA Library Prep Kit (New England Biolabs, USA). Qualitative and quantitative evaluation of the RNA and cDNA libraries was performed with a Qubit fluorometer (Invitrogen, USA) and 2100 BioAnalyzer (Agilent Technologies, USA). The validation of the cDNA libraries before sequencing was performed using a CFX96 Touch Real-Time PCR Detection System (Bio-Rad, USA).