Instrument: Illumina HiSeq 2000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: For ribosome profiling, 50 wild type embryos for each condition were collected at a given 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). For RNA input, total RNA was isolated from 400 ul of the 800 ul of clarified extract before ART- seq Nuclease treatment. Poly-A selection was done according to manufacturer guide- lines (Dynabeads mRNA Purification Kit, Cat no.610.06), and RNA fragmented using the Artseq Ribosome Profiling Kit Mammalian protocol. Both RPF and RNA input fragments were cloned according to the Artseq Ribosome Profiling Kit, Mammalian. The final PCR was carried out with an initial 15 second denaturation at 98 °C, followed by 9-12 cycles of 15 seconds at 98 °C, 5 seconds at 55 °C and extension at 72 °C for 10 s. Reactions were separated on a non-denaturing 8% polyacrylamide TBE gel and DNA fragments of the correct size were extracted and sequenced.