Directional mrna seq sample prep

RNAs were extracted from wild-type, cfp-1, set-2 and sin-3 frozen early stage embryos prepared by hypochlorite treatment of young adults (>95% <200 cell stage). Two to three independent biological replicates were performed for each strain. RNAs were extracted with NucleoZol [Macherey-Nagel] according to manufacturer's instructions and treated with DNAse [Turbo-free DNAse, Ambion]. Integrity of RNA was assessed on Tape Station 4200 [Agilent]. RNA-seq librairies were generated at the GenomEast Platform [IGBMC, Strasbourg, France] using the directional mRNA-Seq SamplePrep [Illumina] and sequenced using the Illumina Hiseq 4000 technology. All RNA-seq data were mapped to the C. elegans reference genome (WS254) by RNA-STAR (Version 2.4.1d). Reads below a mapping score of 10 were filtered using SAMtools (Version 0.1.19). Of the 46 771 annotated genes, 20 183 were selected as protein coding genes and among them, 11 630 had sufficient read representation (baseMean > 10) for further analysis. The gene expression level in each sample was calculated by htseq-count (Version 0.7.2) and differential expression between the different strains was calculated with DESeq2 (54 (link)). Gene expression data are available at GEO with the accession GSE110072.

Poly-adenylated RNA was isolated with Oliogtex (QIAGEN). rRNA was depleted from total RNA with Ribo-Zero Gold (EpiCentre). Libraries were prepared using the Illumina Directional mRNA-Seq Sample Prep and the NEBNext Multiplex Small RNA Library Prep kits and then sequenced on an Illumina HiSeq 2500. RNA-seq reads were filtered for quality and to remove adapters, aligned to hg19 using TopHat2, and transcripts identified with Cufflinks v2.1.1 using default settings.