TAIL-seq in cde-1 mutants versus WT C. elegans infected by the Orsay virus

Aim: We question non-templated transcript 3' tails in cde-1 mutants versus WT C. elegans (N2 strain) infected by the Orsay virus Methods: Approximately 200 cde-1 (tm1021) mutants or N2 animals were infected for 48 hours from the L2 stage to the adult stage with 20 µl filtrate of the Orsay virus on 50mm plate seeded with HB101 bacteria, in biological duplicates. Animals were then washed in M9 and resuspended in 1 ml TRIsure (Bioline). Samples were freeze-thaw three times using liquid nitrogen and RNA purification was performed according to Bioline's guidelines. TAILseq preparation and sequencing was performed as described in Chang, et al. Moll. Cell 2014. Tail-seq libraries were processed using Tailseeker 2 (Chang et al. Moll. Cell 2014). The 5' and 3' libraries were subsequently adapter trimmed using cutadapt 1.10 (Martin. EMBnet.journal 2011) with Illumina small RNA-seq adapters and filtered to a minimum length of 5bp. Trimmed 5' reads were mapped with STAR 2.5.2a (Dobin, et al. Bioinformatics 2013) against a combined meta-genome consisting of the C. elegans reference genome WBcel235 (Harris, et al. Nucleic Acids Res. 2014) and the Orsay virus genome (Felix, et al. Plos Bio. 2011). Mapping was performed in end-to-end mode allowing no mismatches and a gap opening and extension penalty of 10000. Reads were assigned to genes with bedtools 2.26.0 (Quinlan and Hall. Bioinformatics 2010). Subsequently, 3' reads without poly(A) tail or too many dark cycles were removed from the data. For the subsequent analysis, all C. elegans tags with poly(A) tail length equal to zero were discarded. Average poly(A) tail lengths and uridylation lengths for each sample were calculated as the arithmetic mean weighted by the support for each tag, reported by Tailseeker. Overall design: Examination of non-templated tails at the 3' end of transcripts in Orsay virus infected animals from two different genetic backgrounds