Genome wide map of long noncoding RNA Tug1 binding sites in cultured mouse podocytes

We generated a genome wide map of instances where the long noncoding RNA, Tug1, binds to DNA in cultured mouse podocytes under normal glucose conditions using Chromatin-RNA Precipitation coupled with high throughput sequencing (ChIRP-Seq) 48 alternating (even, odd) biotynilated probes were designed to span the full length of Tug1 RNA. Chromatin was prepared from gluteraldehyde crosslinked nuclei from early passage podocytes. Chromatin extracts were duplicated with either even or odd probes. Duplicate samples for Input DNA, Even pulldown (PD) and Odd PD DNA was purified following incubation and supplied for Illumina sequencing by ArrayStar (Rockville, MD).

本研究在正常葡萄糖培养条件下，于培养的小鼠足细胞中构建了长链非编码RNA（long noncoding RNA）Tug1与DNA结合位点的全基因组图谱。实验采用染色质RNA沉淀联合高通量测序（Chromatin-RNA Precipitation coupled with high throughput sequencing，ChIRP-Seq）技术，设计了48条交替排布的生物素标记探针以覆盖Tug1 RNA的全长。染色质样品从早期传代足细胞的戊二醛交联细胞核中提取制备。将染色质提取物分为两组，分别使用正向或反向探针进行重复富集实验。孵育完成后，纯化得到Input DNA（输入对照样本）、正向探针富集（PD）样本及反向探针富集（PD）样本的重复样品，交由位于美国马里兰州罗克维尔市的ArrayStar公司完成Illumina测序。