title: GSM7086457: L4_mut-16_mRNA-20C_rep_1; Caenorhabditis elegans; RNA-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. mRNA-seq library preparation. Nuclease-free H2O was added to 7.5μg of each RNA sample, extracted from whole animals, to a final volume of 100μL. Samples were incubated at 65°C for 2 minutes then incubated on ice. The Dynabeads mRNA Purification Kit (ThermoFisher 61006) was used according to the manufacturer's protocol. 20μL of Dynabeads was used for each sample. 100ng of each mRNA sample was used to prepare libraries with the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB E7760S) according to the manual, using NEBNext multiplex oligos for Illumina (NEB E7335S). Library quality was assessed (Agilent BioAnalyzer Chip) and concentration was determined using the Qubit 1X dsDNA HS Assay kit (ThermoFisher Q33231). 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.