title: Illumina HiSeq 2500 paired end sequencing; Effect of heat stress on developing wheat grain
design: Effect of heat stress on developing wheat grain
library_name: Sample 14_p
library_construction_protocol: Grains were collected from the outside 2 florets (2nd and 4th floret) on the central eight spikelets of the ear of the main tillers, resulting in 16 grains being collected in total from each plant.   Seedlings were grown, individually, in 1.5L pots containing ~950g of a standard soil mix (75% medium grain peat, 12% sterilised loam, 3% medium grade vermiculite 10% sand and 3.5kg Osmocote plus ™ per m³) in a glasshouse with additional overhead lighting set to a 16-hour photoperiod (5.00-21.00), with the canopy receiving ~450 /m2/s Photosynthetically active radiation (PAR). The average relative humidity (RH) of the growing period was 67% until anthesis.Anthesis was defined as the moment when the middle third of the ear of the primary tiller of a plant had anthers protruding.   Four controlled environment growth cabinets (Weiss Technik™ HGC 1514) were used, with two cabinets being used for the high p.a. temperature treatment and two for the control treatment in each experiment. All four cabinets were on a 16-hour photoperiod (5.00-21.00) with night-time conditions of 70% humidity and 15°C. In the high p.a. temperature treatment cabinets, during the light period, humidity was set at 50% whilst the temperature increased from the night-time temperature to 35°C over 4-hours, equating to an increase in temperature of 0.083°C per minute. There was also a ramping of the temperature at the end of each light period, with the temperature decreasing from 35°C to 15°C between 19.00-21.00 hours (a temperature decrease of 0.166°C per minute). Therefore, the high p.a. temperature treatment (35°C) was imposed for 10 hours per day. The two control cabinets were set to the same photoperiod and RH as the high p.a. temperature treatment cabinets for both night and day, although the day/night temperature used in the control cabinets was 18/15°C and there was no ramping of the temperature between night and day. Average amount of light provided was ~550 /m2/s PAR at the canopy level in both sets of cabinets.   For the application of the high p.a. temperature treatments, plants were grouped into differently sized cohorts , with cohorts consisting of plants that had all reached anthesis on the same day.  Total RNA was extracted from freeze-dried material collected at 6, 10 and 14daa in Experiment 2, i.e. samples that had received 0, 4 and 8 days of temperature treatment respectively. Two extractions were performed on grain samples collected at 6daa, and three extractions were performed for each of the two treatments (high temperature/control) at the two further collection points (10 and 14daa) with 14 RNA extractions performed in total. Total RNA was extracted from samples via phenol/chloroform extraction as detailed in Singh et al. Plant Mol. Biol. Rep. 2003;21:93-93. RNA purification and DNA removal was performed prior to resuspension of the RNA pellet using a Qiagen™ RNAse free DNase kit (79254).  Library preparation by Earlham Institute
attributes.tag: Experimental Factor: age
attributes.value: 14 days post anthesis
attributes.tag: Experimental Factor: environmental stress
attributes.value: warm/hot temperature regimen