Illumina HiSeq 1500 sequencing; sRNA-seq of Brachypodium distachyon roots inoculated with Serendipita indica (syn. Piriformospora indica)
Brachypodium distachyon roots were pooled in biological samples (three roots per sample).  Si mycelium and spores were collected from four-week-old axenic cultures grown on CM medium. All samples in triplicates were shock frozen, stored at -80°C and ground in liquid N2. The seeds of Brachypodium distachyon (Bd) line Bd21-3 (gift from R. Sibout, INRA Versailles, France) were surface sterilized (3% active chlorine, sodium hypochlorite solution) for 15 min, washed three times and placed on half-strength MS medium in dark at 4°C for two days and then seven days at 24°C and 16h light / 8h dark cycle (47 μmol m−2 s−1 photon flux density). Serendipita indica (Si) (IPAZ-11827, Institute of Phytopathology, Giessen, Germany) was grown on complete media plates (CM) at 23°C in dark for 4-5 weeks. For inoculation, Si mycelium was collected in 0.002% aqueous Tween 20 solution, filtered (Miracloth, Calbiochem) and pelleted by centrifugation (10 min/4000 rpm/20°C) twice. Chlamydospore concentration of 500,000 conidia ml-1 in 0.002% Tween 20 solution was used to inoculate seven-day-old plant seedlings for 2-3 h. Control plants were mock treated with the 0.002% Tween 20 solution for the same time. Brachypodium distachyon roots inoculated with Serendipita indica or mock-inoculated in Tween water (0.002% Tween 20 solution) were grown on a mixture (2:1, v/v) of vermiculite (Deutsche Vermiculite GmbH) and oil dri (oil binder Typ III R Coarse grain, Damolin, Mettmann, Germany) under 16 h light (160 μmol m-2 s-1, 22°C) and 8h dark (18°C) conditions at 60% relative humidity for four days. Total RNA was isolated using the ZymoBIOMICS RNA Mini Kit (Zymo Research, USA), quantified with DropSense16/Xpose (BIOKÉ, Netherlands) and analyzed with an Agilent 2100 Bioanalyzer Nano Chip (Agilent, Germany). RNA Clean and Concentrator 25 and 5 kits (Zymo Research) were utilized to separate total RNA into fractions: 17-200 nt and > 200 nt. Quantity and quality of the smaller RNA fractions were assessed with the Qubit fluorometer (Invitrogen, Germany) and Agilent 2100 Bioanalyzer Pico Chip. sRNA library preparation was done with 50 ng of RNA (TruSeq Small RNA Library Prep, Illumina) and size selection with the BluePippin (Sage Science, USA) for fragments between 140 and 160 nt (15-35 nt without adapters) applied.