title: C3
organism: Caenorhabditis elegans
description: Protocols: Worms were age-synchronised by egg-prep and were transferred onto experimental plates at the L4 stage. A maximum of 40 worms were transferred per plate for metformin-supplemented plates seeded with OP50-C bacteria, while a maximum of 100 worms were per plate were transferred for the other three conditions. In total, approximately 1000 worms were transferred per condition for 4 independent biological replicates. Worms were then maintained at 20 degreed C before collection on day 2 of adulthood. Worms were collected from plates in sterile nuclease free water and were transferred to 2 mL lysing matrix D tubes (MP Biomedicals) with lysing beads removed. Worms were then washed three times by letting the worms settle at the bottom of the tube, removing the supernatant and resuspending them in sterile nuclease free water. This step was carried out so that any eggs that had been collected with the worms were removed. On the final wash, the supernatant was removed to leave worms in 100 uL of liquid. The lysing beads were added back to tubes with 400 uL of TRIzol and after ten seconds of shaking, samples were flash frozen in liquid nitrogen and were then stored at -80 C until required. Frozen samples were vortexed until thawed and then flash frozen in liquid nitrogen again. Samples were then vortexed for a total of 10 minutes with 30 seconds of chilling on ice every 2 minutes to prevent samples from heat exposure. Samples were transferred to RNAse free tubes and 1 volume of 100% ethanol was added. RNA was extracted using a Direct-zol RNA Miniprep kit (Zymo Research) according to the manufacturers protocol. The RNA concentration of each sample was measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific) and a Quibit assay kit (Thermo Fisher Scientific). The RNA quality of each sample was measured using a 2100 BioAnalyzer (Agilent). Samples were submitted to Genewiz, Inc (NJ, USA) for RNAseq. RNA library preparation and sequencing was performed by Genewiz Inc. RNA libraries were prepared using NEBNext Ultra RNA library preparation kit with poly-A selection.