title: GSM7086464: L4_WT_small-RNA-25C_rep_2; Caenorhabditis elegans; ncRNA-Seq
library_construction_protocol: 10,000 animals per sample were harvested as L4s8,000 animals per sample were harvested as L4s (~56hrs at 20°C and ~39hrs at 25°C) for RNA extraction. Worms were washed off plates using water and then settled on ice to form a pellet. Water was aspirated off and worm pellets were resuspended in 1mL TRIzol reagent (Life Technologies) and freeze-thawed on dry ice followed by vortexing. Worm carcasses were pelleted using centrifugation and the supernatant containing RNA was collected. 0.2 volume chloroform was add to supernatant, vortexed, centrifuged, and then the aqueous phase was transferred to a new tube. Samples were precipitated using isopropanol and rehydrated in 50μL nuclease-free H2O. Small RNA library preparation. Small RNAs (18- to 30- nt) were size selected on denaturing 15% polyacrylamide gels (Criterion 3450091) from total RNA samples. Libraries were prepared as previously described(Montgomery et al., 2012). Library quality was assessed (Agilent BioAnalyzer Chip) and concentration was determined using the Qubit 1X dsDNA HS Assay kit. Libraries were sequenced on the Illumina NextSeq500 (SE 75-bp reads) platform. Two biological replicates were generated for wild-type (N2) and mut-16 mutants cultured at 20°C and 25°C.