Nanopore Transcriptomic Sequencing with C. elegans v1

This protocol entails sequencing native RNA of young adult C. elegans using NanoPore sequencing (Oxford NanoPore Technologies, ONT). RNA extraction will be conducted using modified versions of Zymo’s “Direct-zol‱ RNA Miniprep” (Zymo R2050) and ONT’s “Worm (Caenorhabditis elegans) RNA” protocols. Library preparation will be conducted according to ONT’s Direct RNA Sequencing Kit (ONT SQK-RNA004) protocols and sequencing will be conducted on a MinION Mk1C device (Figure 1). Subsequent analysis should be conducted via alignment and assembly by the wf-transciptomes workflow in EPI2ME followed by followed by differential expression analysis with DESeq2 and functional enrichment with Goseq and KOBAS 2.0 in R Studio.