Paracentrotus lividus Transcriptome or Gene expression

In sea urchin, fertilization triggers an increase in protein synthesis activity and recruitment of stored maternal mRNAs into polysomes. This translational activation is necessary for the onset of the first embryonic cell cycles, for which transcription of zygotic genome is dispensable. To identify mRNAsParacentrotus lividus unfertilized eggs or 1-hour (1-cell) post fertilization embryos were separated by velocity sedimentation through sucrose gradients. Translating mRNAs sediment with polysomes in heavy fractions of the gradient whereas free mRNAs sediment in lighter fractions. Puromycin-treated eggs or 1 cell-embryos were also used to prepare lysates for polysome gradients to determine whether the mRNAs co-sedimenting with the polysomal fractions are actively engaged in translation. In addition to analyzing the samples by polysome profiling, we also used the unfractionated lysates to purify the cytoplasmic RNAs corresponding to each polysome sample fraction. RNA was ext racted from polysomes fractions or from unfractionated lysates using acid phenol/chloroform extraction followed by double precipitation. mRNA-seq samples were prepared from 5 µg of total RNA using the Illumina Truseq mRNA-stranded kit, and sequenced using 100-base length read chemistry in a paired-end flow cell on Illumina HiSeq 2000. RNAseq data were generated from three independent experiments, comprising eight different samples as follow: cytoplasm and polysomal fractions, in eggs and 1-cell embryos, and in absence or presence of puromycin.