Illumina HiSeq 2500 sequencing; RNA-seq  of Caenorhabditis elegans at Late L4 stage of WT, daf-2(e1370), daf-16(mgDf50);daf-2(e1370) on control RNAi and cdk-12 RNAi
RNA-seq  of Caenorhabditis elegans at Late L4 stage of WT, daf-2(e1370), daf-16(mgDf50);daf-2(e1370) on control RNAi and cdk-12 RNAi
Worms grown on control or cdk-12 RNAi were collected using 1XM9 buffer and washed thrice to remove bacterial contamination. 200 l of Trizol reagent (Takara, Japan) was added to the 50l of the worm pellet and subjected to three freeze-thaw cycles in liquid nitrogen with intermittent vertexing to break open worm bodies. The samples were then frozen in liquid nitrogen and stored in -80 C till further use. 200 l of Trizol was again added to the worm pellet and the sample was vortexed vigorously. To this, 200 l 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 4C. 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 4C. 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 4C. RNA pellet was further dried at room temperature and later dissolved in DEPC (diethyl pyrocarbonate) treated water followed by incubation at 65C 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.  Libraries were constructed using NEBNext Poly(A) mRNA Magnetic Isolation Module (Catalog no-E7490L) and NEBNext Ultra II Directional RNA Library Prep kit (Catalog no-E7765L) according to the manufacturers instructions.