Deep-transcriptome and ribonome sequencing redefines the molecular networks of pluripotency and the extracellular space in human Embryonic Stem cells

Cytoplasmic polyadenylated RNA from human embryonic stem cells (HES2) was separated on a 1.2% agarose and gel slices were excised corresponding to the following sizes: 0 to 0.5 kb; 0.5-2 kb; 2-3.5 kb; 3.5-6.5 kb and 6.5-20+ kb. Slices were dissolved and extracted RNA (1% of each fraction) was run on the Agilent Bioanalyser (Agilent) to confirm the correct size distribution and yield. Library molecules were clonally amplified onto one micron magnetic beads according to the SOLiD Template Bead Preparation protocol and sequenced using a SOLiD Analyzer according to manufacturer's instructions. Overall design: Human ES cells size fractionated mRNA

从人类胚胎干细胞（HES2）中提取的胞质多聚腺苷酸化RNA经1.2%琼脂糖凝胶电泳分离后，切取对应以下分子量范围的凝胶切片：0~0.5 kb、0.5~2 kb、2~3.5 kb、3.5~6.5 kb及6.5~20+ kb。将凝胶切片溶解并提取RNA，取各组分的1%通过安捷伦生物分析仪（Agilent Bioanalyser，安捷伦）进行检测，以确认RNA的分子量分布与产出量符合预期。依照SOLiD模板磁珠制备方案，将文库分子克隆扩增至1微米磁珠表面，随后按照制造商操作指南，使用SOLiD测序分析仪完成测序。实验整体设计：经分子量分级分离的人类胚胎干细胞mRNA