BGISEQ-500 paired end sequencing; Transcript Profiling after a Genetically-induced Elevation of Trehalose 6-Phosphate Reveals a Plethora of Direct and Indirect Responses
Seeds were sown on a 1:1 mixture of compost (Stender AG, Schermbeck, Germany; https://www.stender.de) and vermiculite in 6-cm diameter pots, covered and kept at 4°C in the dark for 48 h then transferred to a controlled environment chamber (Percival E-36 L chamber, CLF Plant Climatics GmbH, Weringen, Germany; https://www.plantclimatics.de/) with a 16-h photoperiod (160 µmol m-2 s-1 irradiance provided by white LEDs) and day/night temperatures of 21°C/19°C. After germination, seedlings were transferred to 10-cm diameter pots (five seedlings per pot), and grown as above until 21 days after sowing (DAS). Three-weeks-old plants were sprayed to run-off with water (mock-induced control) or 2% (v/v) ethanol (induced) at the beginning of the day (ZT0.5) and harvested at time 0 (in the light, before spray), 2, 4 and 6 h after spraying. Total RNA was extracted with Plant RNA easy extraction kit according to manufactures protocol. RNA concentration and purity were estimated by photometric analysis using a NanoDrop® ND-1000 UV-VIS spectrophotometer (Thermo Fisher Scientific Inc. Waltham, MA., USA; www.thermoscientific.com). If necessary, RNA was diluted with water to a final concentration of 2 µg/µl. RNA integrity was confirmed by agarose (1%) gel electrophoresis and by Bioanalyzer 2100 (Agilent Technologies). DNA was removed by treatment with Turbo DNA-free™ DNase I enzyme (Applied Biosystems) according to the manufacturer's instructions. This was done by BGI genomics. Quality control and the processing of raw data were done by BGI and included barcode trimming and the removal of adaptor sequences, as well as of low-quality reads, generating 20M clean Pair Ends reads (100 bp) for each sample.