GSM3589996: mutant_starved_rna1; Caenorhabditis elegans; RNA-Seq
Worms were collected in Eppendorf tubes and washed three times in M9.  We added 400µL of TRIzol (Thermoscientific, 15596026) to the worm pellet (containing ~50,000 worms/sample), and the tube was frozen immediately in liquid nitrogen and stored at -80°C.  Following three cycles of freeze-thaw, 200µL of TRIzol was added with incubation at room temperature for five minutes. Thereafter, 140µL of chloroform (Sigma, 319988) was added, the samples were shaken by hand to mix for 60 seconds and incubated at room temperature for two minutes; followed by centrifugation at 12,000 X g for 15 minutes at 4°C.  The aqueous phase and an equal volume of 70% ethanol were added to a fresh 1.7mL tube.  Nucleic acids were isolated with the RNeasy Mini Kit (Qiagen, 74104) following manufacturer's instructions. Illumina TruSeq