RNA-Seq Analysis in hES/ iPS cell-derived neuronal samples

We characterized the gene expression by Hierarchical Clustering and one-matrix clustering in hESC, day 12 progenitors, day 25-day 27, day82 differentiated hypothalamic neurons from hESCs and day 45 neurons derived from iPSCs generated from controls (2 independent) and BBS (Bardet-Biedl Syndrome, 3 independent) subjects. RNA was isolated from cells of 13 samples (1 hESC, triplicate for day 12 progenitors, 1 day 25 neuron sample, duplicate for day 27 neuron samples, 1 day 82 neuron sample, five day 45 neuron samples made from 5 independent iPSC lines ) using RNeasy Micro Kit (QIAGEN). Quality control of the RNA was carried out with the Agilent Bio-analyzer, Qubit 2.0 at the MPSR of Columbia University. 100 ng of RNA with RIN ≥ 9 were used for generating mRNA-focused libraries using TruSeq RNA Sample Preparation Kit v2 and sequencing on an Illumina 2000/2500 V3 Instrument offered by the Columbia Genome Center.

本研究通过层次聚类（Hierarchical Clustering）与单矩阵聚类（one-matrix clustering），对以下样本的基因表达特征进行了表征：人类胚胎干细胞（human embryonic stem cells, hESC）、第12天祖细胞、第25至27天神经元、源自hESC的第82天分化下丘脑神经元，以及源自对照个体（2株独立样本）和巴德-毕德氏综合征（Bardet-Biedl Syndrome, BBS）患者（3株独立样本）的诱导多能干细胞（induced pluripotent stem cells, iPSCs）所制备的第45天神经元。本研究使用RNeasy微量试剂盒（QIAGEN），从13份样本的细胞中提取总RNA，具体样本包括：1份hESC样本、第12天祖细胞的3次生物学重复样本、1份第25天神经元样本、第27天神经元样本的2次生物学重复样本、1份第82天神经元样本，以及由5株独立iPSC系制备的5份第45天神经元样本。本研究在哥伦比亚大学分子病理学与标本资源库（MPSR），采用安捷伦生物分析仪与Qubit 2.0荧光定量仪完成RNA质量控制。选取RNA完整性数（RIN）≥9的100 ng RNA，使用TruSeq RNA样本制备试剂盒v2构建靶向mRNA的文库，并依托哥伦比亚大学基因组中心提供的Illumina 2000/2500 V3测序仪进行测序。