mRNA-seq analysis of sir-2.1 mutants

mRNA-seq analysis of sir-2.1 null mutant strain VC199 (sir-2.1) was performed as described previously and compared to previously published mRNA-seq data (Kramer et al., 2015 (link)). Briefly, embryos and L2/L3 larvae were collected for at least three biological replicates. After collection, worms were stored in Trizol (Invitrogen). RNA was purified following the manufacturer's protocol after freeze-cracking samples five times. RNA was cleaned up using a Qiagen RNeasy kit, and mRNA was purified using Sera-Mag Oligo (dT) beads (Thermo Fisher Scientific) from 1 µg of total RNA. Stranded Illumina libraries were prepared as described previously (Kramer et al., 2015 (link)), and sequencing was performed with an Illumina HiSeq-2000 to produce single-end 50–75 bp reads. We aligned reads to the WS220 genome version using Tophat version 2.1.1 with default parameters (Kim et al., 2013 (link)). Count data was calculated using HTSeq version 0.6.1 (Anders et al., 2015 (link)) and normalized using the R package DESeq2 (Love et al., 2014 (link)). TPM counts and DEseq output files are provided in Tables S9–S14.