Illumina HiSeq 4000 sequencing; Cross-species screening platforms identify EPS-8 as a critical link for mitochondrial stress and actin stabilization.
Cross-species screening platforms identify EPS-8 as a critical link for mitochondrial stress and actin stabilization.
Animals were synchronized using a standard bleaching protocol and all RNA collection was performed at day 1 of adulthood. ~1000 animals were harvested using M9, and animals were pelleted by gravity by allowing worms to settle to the bottom of the tube over 2 minutes. M9 was subsequently aspirated and replaced with Trizol solution, and worms were freeze/thawed 3x with liquid nitrogen, and a ~30 sec vortexing performed before each re-freeze.  M9 was subsequently aspirated and replaced with Trizol solution, and worms were freeze/thawed 3x with liquid nitrogen, and a ~30 sec vortexing performed before each re-freeze. After the final thaw, chloroform was added at a 1:5 ratio (chloroform:trizol) and aqueous separation of RNA was performed via centrifugation in a heavy gel phase-lock tube (VWR, 10847--802). The aqueous phase was collected, mixed with isopropanol at a 1:1 ratio, then RNA purification was performed using a Qiagen RNeasy Mini Kit (74106) as per manufacturerâ€™s directions.  For RNAseq analysis, library preparation was performed using Kapa Biosystems mRNA Hyper Prep Kit. Sequencing was performed at the Vincent J. Coates Genomic Sequencing Core at the University of California, Berkeley using an Illumina HS4000, mode SR100.  RNA concentration from 3 biological replicates was measured using an Agilent Bioanalyzer at UC Berkeley Functional Genomics Core Lab (FGL). Total RNA (500 ng) was used for library construction using the mRNA HyperPrep Kit (KAPA Biosystems KR1352).
Experimental Factor: organism
Caenorhabditis elegans
Experimental Factor: RNA interference
fed on empty vector control
Experimental Factor: genetic modification
none