GSM8706138: N2 C elegans, control, embryo stage, rep1; Caenorhabditis elegans; RNA-Seq
Age-synchronised worms from a single NGM plate were incubated with TRIZol (500 ml per NGM plate), samples were mixed by vortexing for 30 sec. Chloroform was added to each tube (100 μL for a 12-well plate and 1 ml for a C. elegans 60 mm Ø plate), and the tube shaken vigorously by hand for 15 sec before incubation for 5 min at room temperature. RNA precipitation was achieved by adding 100% isopropanol to the aqueous phase (250 μL for a 12-well plate) and incubating at -20ºC overnight. RNA pellet was washed by adding 100 μL of 80% ethanol, supernatant was discarded and the tubes were allowed to air dry for 5-10 min at room temperature. The RNA was reconstituted in 10 μL of RNase-free H2O. Samples were analysed using the NanoDrop2000 spectrophotometer to quantify nucleic acid content and purity. Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers, followed by the second strand cDNA synthesis using either dUTP for directional library or dTTP for non-directional library. For the non-directional library, it was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. For the directional library, it was ready after end repair, A-tailing, adapter ligation, size selection, USER enzyme digestion, amplification, and purification. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries will be pooled and sequenced on Illumina platforms, according to effective library concentration and data amount.