Transcriptomic analysis of mitohormesis associated with lifespan extension in Caenorhabditis elegans
Transcriptomic analysis of mitohormesis associated with lifespan extension in Caenorhabditis elegans
For RNA isolation, all RNa collection was performed at day 2 of adulthood. ~1000 animals were harvested from RNAi plates using M9. Animals were pelleted by gravity by allowing adult worms to settle to the bottom of the tube and aspirating off eggs and L1. Animals were washed and gravity settled 3x to remove a majority of progeny, then animals were placed into Trizol solution and worms were freeze/thawed 3x with liquid nitrogen with a 30 sec vortexing step between each freeze cycle.  Standard bleaching protocol was used to synchronize animal populations. Animals were L1 arrested for a maximum of 16 hours to further synchronize populations. Animals were grown on RNAi indicated from L1 until Day 1 of adulthood and material was collected on Day 1.  After the final thaw, chloroform was added at a 1:5 chlorform/trizol ratio and aqueous separation of RNA was performed via centrifugation in a heavy gel phase-lock tube (VWR, 10847-802). The aqueous phase was mixed 1:1 with isopropanol then applied to a Qiagen RNeasy Mini Kit (74106) and RNA purification was performed as per manufacturerâ€™s directions.  Library construction was performed by Novogene using their standard pipeline.