RNA-seq of Caenorhabditis elegans (at Late L4 stage) of rrf-3(pk1426);rde-1(ne219);unc-62p::rde-1 and daf-2(e1370);rrf-3(pk1426);rde-1(ne219);unc-62p::rde-1 grown on control RNAi or cyd-1 RNAi.
RNA-seq of Caenorhabditis elegans (at Late L4 stage) of rrf-3(pk1426);rde-1(ne219);unc-62p::rde-1 and daf-2(e1370);rrf-3(pk1426);rde-1(ne219);unc-62p::rde-1 grown on control RNAi or cyd-1 RNAi.
Worms grown on control or cyd-1 RNAi were collected using 1XM9 buffer and washed thrice to remove bacterial contamination. 200 μl of Trizol reagent (Takara, Japan) was added to the 50μl of the worm pellet and subjected to three freeze-thaw cycles in liquid nitrogen with intermittent vortexing to break open worm bodies. The samples were then frozen in liquid nitrogen and stored in -80 °C till further use. 200 μl of Trizol was again added to the worm pellet and the sample was vortexed vigorously. To this, 200 μl of chloroform was added and tube was gently inverted several times followed by 3 minutes incubation at room temperature. Sample was then centrifuged at 12000g for 15 minutes at 4°C. The RNA containing upper aqueous phase was gently removed into a fresh tube without disturbing the bottom layer and interphase. To this aqueous solution, an equal volume of isopropanol was added and the reaction was allowed to sit for 10 minutes at room temperature followed by centrifugation at 12000g for 10 minutes at 4°C. The supernatant was carefully discarded without disturbing the RNA containing pellet. The pellet was washed using 1 ml 70% ethanol solution followed by centrifugation at 7500g for 15 minutes at 4°C. RNA pellet was further dried at room temperature and later dissolved in DEPC (diethyl pyrocarbonate) treated water followed by incubation at 65°C for 10 minutes with intermittent tapping. Concentration of RNA was determined by measuring absorbance at 260 nm using Nano Drop UV spectrophotometer (Thermo Scientific, USA) and quality checked using denaturing agarose formaldehyde gel.  1.5 ug of RNA samples was used for polyA mRNA isolation from total RNA using NEBNext® Poly(A) mRNA Magnetic Isolation Module (Catalog no-E7490L) according to the maufacturer's protocol.The NEBNext® Ultra™ II Directional RNA Library Prep kit (Catalog no-E7760L) was used to construct libraries according to the manufacturer's instructions.
European Bioinformatics Institute
E-MTAB-13172:daf-2;rrf-3;rde-1;unc-62p::rde-1 cyd-1 RNAi_Rep1_p