Paracentrotus lividus Transcriptome or Gene expression. Paracentrotus lividus

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.在海胆（sea urchin）中，受精会触发蛋白质合成活性的提升，并将储存的母源mRNA（maternal mRNAs）招募至多聚核糖体（polysomes）中。这种翻译激活对于首次胚胎细胞周期的启动是必需的，而合子基因组（zygotic genome）的转录在此阶段并非必需。为了鉴定副紧海胆（Paracentrotus lividus）未受精卵子及受精后1小时（1细胞期）胚胎中的mRNA，我们通过蔗糖梯度速度沉降对样本进行了分离。正在翻译的mRNA会与多聚核糖体结合，沉降于梯度的重组分中，而游离mRNA则沉降于轻组分中。我们还使用了经嘌呤霉素（puromycin）处理的卵子及1细胞期胚胎制备裂解液，用于多聚核糖体梯度实验，以确定与多聚核糖体组分共沉降的mRNA是否正活跃地参与翻译过程。除了通过多聚核糖体谱分析样本之外，我们还利用未分级的裂解液纯化了对应每个多聚核糖体样本组分的细胞质RNA。采用酸性苯酚/氯仿抽提法，随后进行两次沉淀，从多聚核糖体组分或未分级裂解液中提取RNA。mRNA测序（mRNA-seq）样本以5μg总RNA为起始材料，使用Illumina Truseq mRNA-stranded试剂盒制备，并在Illumina HiSeq 2000的双端流动槽中采用100碱基读长测序化学进行测序。RNA测序数据来自3次独立实验，共包含8种不同样本，具体如下：卵子和1细胞期胚胎的细胞质组分与多聚核糖体组分，以及存在或不存在嘌呤霉素的条件下的对应样本。