title: Illumina Genome Analyzer IIx sequencing; Comparative transcriptome profiling of the response to Pyrenochaeta lycopersici in resistant tomato cultivar Mogeor and its background genotype - susceptible Moneymaker.
design: Comparative transcriptome profiling of the response to Pyrenochaeta lycopersici in resistant tomato cultivar Mogeor and its background genotype - susceptible Moneymaker.
library_construction_protocol: Tomato plants were grown in greenhouse at 28 °C. Young tomato plants (3rd leaf stage) were inoculated by dipping the roots for a few seconds into a homogenate of the fungus grown on PDA (Potato Dextrose Agar), then transplanted in pots containing sterile vermiculite and maintained in greenhouse until evaluation. The roots of control plants were dipped into a PDA homogenate following the same protocol used for inoculation with the fungus. Three independent biological replicates, each consisting of 5 plants, were performed for both control and infected with P. lycopersici MM and M, for a total of 60 plants.RNA extraction Total RNA was extracted 96 hours post-inoculation (hpi).  Total RNA was extracted from 100 mg of roots from P. lycopersici infected and healthy MM and M plants. The samples were homogenized in liquid N2 and RNA was isolated using the NucleoSpin RNA plant kit (Macherey-Nagel,  Düren, Germany) according to the manufacturer's instructions, except for the digestion on column with rDNAse, for which the incubation time was extended to 30 min. RNA concentration and purity/integrity were analysed using first NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and then Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA ).  RNA-Seq libraries were prepared with the TruSeq RNA sample preparation kit (Illumina), according to manufacturers' instructions, starting from 1 µg of total RNA for each sample. This protocol involves removal of rRNA. The quality of libraries was checked with a 2100 Bioanalyzer (Agilent) using the Agilent DNA 1000 Kit and following provided instructions. Libraries were quantified through qRT-PCR, as recommended by the manufacturers' instructions, and single-end sequenced for 51 bases on an Illumina Genome Analyzer (GAIIx).