title: RNA-Seq of mRNA derived from fourth larval stage larvae from C. elegans strain PE1328 treated for 16 hours at 20 degrees Celsius with 5-Ph-IAA compared to untreated controls
design: RNA-Seq of mRNA derived from fourth larval stage larvae from C. elegans strain PE1328 treated for 16 hours at 20 degrees Celsius with 5-Ph-IAA compared to untreated controls
library_construction_protocol: Worms were washed off NGM plates using M9 and washed 3 times by centrifugation at 749g for 2 min at 4°C.  1 ml of Trizol (TRI Reagent, T9424, Sigma Aldrich) was added per 50 µl of worm pellet and samples frozen in liquid N2. Samples were thawed at 37°C and re-frozen four times. They were then vortexed for 30 seconds, left to rest for 30 seconds and the vortex/rest process repeated for a further three times. Phase separation by addition of chloroform and recovery of RNA was done as described by the manufacturer.  The aqueous fraction was transferred to a low DNA-binding-tube and diluted 1:1 with 70% ethanol. RNA was purified using PureLink RNA Mini Kit (Thermo Fisher Scientific), using the on-column DNase-treatment option.  Directional library preparation, with rRNA removal was performed (Novogene UK Ltd).