RNA from Panagrolaimus davidi held at -10 degrees for 24 hours, then thawed for 24 hours
For the Illumina sequencing the nematodes were cultured in S medium at 20°C for 3 weeks and fed every 3-4 days with Escherichia coli. After acclimating at +5°C for 3 days, the samples were cooled to -10°C at 0.5°C min-1 and held for 24 h. After which they were warmed to +1°C at 0.5°C min-1 and then kept at 5°C for 24 h. Nematodes were extracted using a modified Baermann technique. The worms were snap frozen in 1.5ml microcentrifuge tubes at -80 degrees Celsius and preserved at -80 degrees Celsius until extraction. RNA was extracted from whole worms using TRI-sure (Bioline) according to manufacturer's instructions and purified on Qiagen RNeasy columns. RNA were checked for purity on standard agarose gels and quantified using a NanoDrop spectrophotometer. 5ug from 8 different treatment stages was sent for sequencing.