Name: Root-short-term-salt-replicate-2_p
Instrument: Illumina HiSeq 2000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: PolyA
Layout: PAIRED
Construction protocol: Three individual plants were used per treatment as biological replicates. At sampling stage the plants had 15-17 stem internodes. The first two fully expanded leaves stem (internodes 5-7) and the corresponding stem internodes were selected for sampling. The major veins were removed from leaves and flash frozen in liquid nitrogen. The bark was peeled off from stem internodes and the stem xylem was split tangentially and flash frozen in liquid nitrogen. Fibrous roots (diameter less than 2mm) were subjected to a quick pressure wash with water to remove soil and flash frozen in liquid nitrogen. All samples were stored at -80 degrees C before RNA extraction. In summary, the three tissues sampled were: (1) mature leaf, (2) stem xylem and (3) root P. trichocarpa Nisqually-1 cuttings (2 inches long) obtained from approximately 12 year old field grown trees (Steve Strauss – Forest Ecosystem and Society field plantation in Corvallis, OR) were used to establish plants in 1 gallon pots containing Sungro Horticulture MM840 soil mix (http://www.sungro.com), under greenhouse conditions during late spring and summer of 2013. The plants were watered every 2 days to field capacity and fertilized weekly (20-20-20, N-P-K Ferti-lome soluble plant fertilizer, 1.5g per L, 300ml per plant). The plants were grown in the greenhouse for 8 weeks before being transferred to a growth chamber (12 h day/night cycle, maximum light intensity of 300 µmol m−2 s−1, temperature of 27 °C and 60% relative humidity). The plants were acclimated under growth chamber conditions for 2 weeks prior to treatments. Control: Plants were watered every 2 to 3 days. Growth chamber conditions (12 h day/night cycle, maximum light intensity of 300 µmol m−2 s−1, temperature of 27 °C and 60% relative humidity). Sampling was carried out 24 hours after watering at mid light period (6-7h after the beginning of light period). Short-term drought: Water deficit conditions were imposed on plants by withholding water. Growth chamber conditions (12 h day/night cycle, maximum light intensity of 300 µmol m−2 s−1, temperature of 27 °C and 60% relative humidity). Sampling was carried out at initial signs of wilting (volumetric soil water content 0.02-0.03 m3/m3, 5 days after withholding water) at mid light period (6-7h after the beginning of the light period). Prolonged drought: After initial wilting (volumetric soil water content 0.02-0.03 m3/m3, 5 days after withholding water), the soil moisture was maintained at 0.02-0.03 m3/m3 by limited watering (3 to 4 times a day 25-50 ml) for 7 days. During this period the plants went through wilting cycles but were not allowed to wilt to an extent to sustain any leaf damage. Sampling was carried out 12 days after withholding water (7 days after initial wilting), at mid light period (6-7h after the beginning of light period). Short-term heat: Heat stress was imposed by increasing the temperature to 39 degrees C, starting mid dark period (6h after the beginning of the dark period). Growth chamber conditions (12 h day/night cycle, maximum light intensity of 300 µmol m−2 s−1, temperature of 39 °C and 60% relative humidity). Sampling was carried out 12h after increasing the temperature at mid light period (6h after the beginning of the light period). Prolonged heat: Heat stress was imposed by increasing the temperature to 39 degrees C mid light period (6h after the beginning of the light period) and maintained at 39 degrees C for 7 days. Growth chamber conditions (12 h day/night cycle, maximum light intensity of 300 µmol m−2 s−1, temperature of 39 °C and 60% relative humidity). Sampling was carried out 7 days after increasing the temperature, at mid light period (6h after the beginning of the light period). Prolonged salt: Plants were watered to field capacity and salinity stress was imposed by adding 400ml of 100mM NaCl to the soil at mid light period (6h after the beginning of the light period). The pots were placed in pot saucers to hold the excess salt solution). Growth chamber conditions (12 h day/night cycle, maximum light intensity of 300 µmol m−2 s−1, temperature of 27 °C and 60% relative humidity). The plants were maintained for 7 days with replenishment of 100mM NaCl in the pot saucer every 2 days. Sampling was carried out 7 days after imposing salt stress, at mid light period (6h after the beginning of the light period). Short-term cold: Cold stress was imposed by reducing the temperature to 12 degrees C during the light period and 4 degrees C during the dark period and. Growth chamber conditions (12 h day/night cycle, maximum light intensity of 300 µmol m−2 s−1, temperature of 4°C night/12°C day, and 60% relative humidity). Sampling was carried out 24h after reducing the temperature, at mid light period (6h after the beginning of the light period). Prolonged cold: Cold stress was imposed by reducing the temperature to 12 degrees C during the light period and 4 degrees C during the dark period and. Growth chamber conditions (12 h day/night cycle, maximum light intensity of 300 µmol m−2 s−1, temperature of 4°C night/12°C day, and 60% relative humidity). Sampling was carried out 7 days after reducing the temperature, at mid light period (6h after the beginning of the light period). Tissue was ground to a powder in liquid nitrogen and suspended in 'PureLink Plant RNA Reagent' (Thermo Fisher Cat # 12322012) and total RNA was extracted following manufacturers protocol. The RNA was treated with DNAse using a TURBO DNA-free kit Reagent' (Thermo Fisher Cat # AM1907), following manufacturer's instructions. The DNA free RNA was purified using a Qiagen RNeasy mini kit following manufacturer's protocol. The quality of RNA was evaluated using a NanoDrop spectrophotometer and an Agilent Bioanalyzer instrument. RNA-seq libraries were prepared using True-Seq kit (Illumina Inc.) according to the manufacture's protocol.