title: Illumina HiSeq 2500 sequencing; 3'UTR sequencing of C.elegans strains
design: 3'UTR sequencing of C.elegans strains
library_construction_protocol: All crosses were done by plating 3-5 hermaphrodites with 10-15 males on petri dishes filled with NGM containing a bacterial lawn. Approximately 5 F1 hermaphrodites at the L4 stage of development were then plated onto plates. F2 progeny were collected at the L4 stage of development and singled out onto separate plates. F2 worms were incubated until they had laid eggs, then collected and lysed. The worm lysate was used for PCR reactions to confirm the presence of the desired genetic lesion.    RNA was extracted from L4 stage worms using TRIzol (Invitrogen).  To construct RNA-seq libraries from C.elegans samples, we used an automated QuantSeq 3'mRNA-seq (Lexogen GmbH, Vienna) using the Agilent NGS Workstation (Agilent Technologies, Santa Clara) as per manufacturer's protocol.  Briefly, 250 ng of total RNA was used to generate cDNA.  cDNA was amplified with 17 PCR cycles as determined by qPCR analysis using the PCR Add-on kit (Lexogen). ERCC RNA Spike-ins were added following manufacturer's instructions. The resulting libraries were quantified with Qubit DNA HS (Thermo Fisher, Waltham) and fragment sizes analyzed on the Agilent Bioanalyzer using the High Sensitivity DNA assay prior to sequencing. 