Plant material, plate set-up and growth conditions
Seed for the 192 Arabidopsis accessions were gas sterilised using
3ml hydrochloric acid in 100ml sodium hypochlorite. We then stratified
seed in 1ml of sterile water for 2 days at 4°C before plating. Clear 96
well plates with flat bottomed wells (Thermo Scientific, cat no:
10287631) were filled with 250ul of Murashige and Skoog agar media
(1.5% Agar, pH5.8, no sucrose). Approximately 20 seeds were added to
each well of the plates following a randomized Alpha-lattice design (see
Extended Methods) and left to dry until all residual water had
evaporated. Each accession was replicated 18 times across the 3 imaging
runs. Clear microplate lids (Thermo Scientific, cat no: 10334311) were
secured to each plate with micropore tape ensuring that the lid was
raised slightly from the top of the wells to allow condensation to
circulate freely. Plates were returned to the fridge for 2 more days
before transfer to the growth cabinet set at 12:12 L:D cycle at 22°C
under approximately 200 µmol m-2 s-1white light for 14 days. Prior to the temperature response experiments,
accessions from the 191 phenotyping screen were bulked for seed (see
Extended Methods). Plates were all grown under 12:12 L:D at 22°C light
for 10 days and were then transferred to the temperatures in which they
would be imaged for 4 days.
Note: For the temperature and mutant screening experiments, the corner
wells of the 96-well plates were not used in the designs as we found
that corners had significant effects on circadian rhythms in the 191
dataset; potentially due to these wells drying out more quickly
(Supplementary Figure 8).