RNA Extraction and Sequencing for C. elegans

RNA was isolated from FACS-sorted samples as previously described [2 (link)]. Briefly, RNA was extracted using standard Trizol/ chloroform/ isopropanol method, DNase digested, and cleaned using Qiagen RNEasy Minelute columns. Agilent Bioanalyzer RNA Pico chips were used to assess quality and quantity of isolated RNA. 10 to 100 ng of the isolated quality assessed RNA was then amplified using the Nugen Ovation RNAseq v2 kit, as per manufacturer suggested practices. The resultant cDNA was then sheared to an average size of ~200 bp using Covaris E220. Libraries were prepared using either Nugen Encore NGS Library System or the Illumina TruSeq DNA Sample Prep, 1 μg of amplified cDNA was used as input. RNA from a subset of samples was amplified using the SMARTer Stranded Total RNA kit-pico input mammalian, as per manufacturer suggested practices. No differences were observed between the two methods, and samples amplified by different methods clustered well (Fig 1B). The resultant sequencing libraries were then submitted for sequencing on the Illumina HiSeq 2000 platform. 35–200 million reads (average of 107,674,388 reads) were obtained for each sample and mapped to the C. elegans genome. Sequences are deposited at NCBI BioProject PRJNA400796.