GSM6413339: UA44 strain, P0 generation, OP50 diet, rep3; Caenorhabditis elegans; RNA-Seq
RNA was extracred by freezing and thawing samples in 500μL of Trizol 7 times, then vortexed and iced 7 times. Phases were separated with 100 μL choloform. After separation, the aqueous phase was added to an equal volume of 70% ethanol and subjected to the protocol outlined in the Qiagen RNeasy Micro Kit. RNA was eluted with 35μL of RNase-free water. mRNA was enriched using oligo(dT) beads and then fragmented randomly in fragmentation buffer, followed by cDNA synthesis using random hexamers and reverse transcriptase. Then, a custom second-strand synthesis buffer (Illumina) was added with dNTPs, RNase H and Escherichia coli polymerase I. The final cDNA library was ready after a round of purification, terminal repair, A-tailing, ligation of sequencing adapters, size selection and PCR enrichment.