title: GSM1152462: MZpou5f1 Nanog + SoxB1 MO 6hpf Total mRNA; Danio rerio; RNA-Seq
library_construction_protocol: For mRNA-Seq, total RNA from five embryos was extracted using Trizol (Invitrogen) for each experimental condition. RNA was treated with TURBO DNase (Ambion) for 30 minutes at 37°C and extracted using phenol chloroform. For ribosome profiling, 50 wild type embryos for each condition were collected at 64-cell stage. Embryos were lysed using 800ul of a mammalian cell lysis buffer containing 100ug/ml Cycloheximide as per the manufacturer’s instruction (ARTseq Ribosome Profiling Kit, RPHMR12126, Epicentre). For nuclease treatment, 3ul of ARTseq Nuclease was used. Ribosome protected fragments were run and 28-29nt fragments were gel purified as previously described in (Bazzini et al., 2012) and cloned according to the manufacturers protocol (ARTseq Ribosome Profiling Kit, RPHMR12126, Epicentre). TruSeq strand-specific libraries were constructed according to standard protocols. For total mRNA-Seq, sequencing libraries were treated with Epicentre Ribo-Zero Gold kits according to the published protocol, in order to deplete ribosomal RNA prior to sequencing. Ribosome profiling libraries were constructed as in (Bazzini et al., Science 2012).