RNA-seq Library Preparation and Analysis

Worms were collected and stored in ten volumes of Trizol (Invitrogen). Samples were freeze-cracked five times and RNA purification was done according to the manufacturer protocol. Isolated RNA was cleaned up using the Qiagen RNeasy kit. The mRNA was purified using Sera-Mag Oligo (dT) beads (Thermo Scientific) from at least 1 ug of total RNA. Stranded mRNA-seq libraries were prepared based on incorporation of dUTPs during cDNA synthesis using a previously described protocol (Parkhomchuk et al., 2009 (link)). Single-end 50 bp sequencing was performed using the Illumina HiSeq-2000. Reads were aligned to genome version WS220 with Tophat version 2.0.0 (Trapnell et al., 2012 (link)) using default parameters. For each biological replicate, read numbers and mapping percentages (which refer to the percentage of unique reads with at least one alignment) can be found in Supplementary file 1B. Count data was calculated using HTSeq version 0.6.1 (Anders et al., 2015 (link)) and normalized using the R package DESeq (Anders and Huber, 2010 (link)). The raw reads and counts can be obtained from GEO under accession number [GEO: GSE87741].