title: Sample 75
organism: Lupinus angustifolius
description: Protocols: Leaves were sampled at 6, 12, 24, 36, and 48 hours post inoculation. Experiment was performed in controlled environment (a greenhouse), in temperature regime 21 °C day / 19 °C night and under 14 h photoperiod. Seeds were scarified before sowing to brake seed dormancy resulting from hard seed coat and provide even germination. Pots (11 cm × 11 cm × 21 cm) with sterilized soil were used. The inoculation was done using Colletotrichum lupini strain Col-08, obtained in 1999 from the stem of narrow-leafed lupin plants cultivated on field in Wierzenica (52°27′42″N 17°04′05″E) in Great Poland region. The isolate was cultured on SNA medium for 21 days at 20°C under black light to induce sporulation. The inoculation was performed 4 weeks after sowing, when the plants reached 4-6 leaf stage, by spraying of conidial spore suspension in concentration 0,5 x 106 conidia per ml. After inoculation, plants were kept for 24 hours in darkness under ~100% humidity and temperature of 25°C to facilitate conidia germination and infection process. Afterwards, plants were grown under 14-h photoperiod in temperature regime 22°C day / 19°C night and humidity ~70%. Frozen leaf tissue (50 mg) was homogenized in 2 ml tubes (Eppendorf, Hamburg, Germany) and two stainless steel beads (ø 5 mm) using TissueLyser II (Qiagen). SV Total RNA Isolation System (Promega, Madison, WI, USA) was used for RNA isolation without any changes to the protocol except extending DNAse I digestion to 25 min. RNA purity and integrity of the samples prepared for sequencing were measured using an Experion™ Automated Electrophoresis System (Bio-Rad, Hercules, CA, USA). TruSeq RNA Sample Preparation v2 Guide, Part # 15026495 Rev. F