RNA-seq analysis of hilar and peripheral biliary organoids from a mouse model of Alagille syndrome

To study molecular differences in wild-type and regenrated bile ducts in a mouse model of Alagille syndrome (Andersson, Chivukula, Hankeova, et al. Gastroenterology, 2018;154(4):1080-1095), we derived intrahepatic cholangiocyte organoids from hilar (pICOs) and peripheral (pICOs) regions of adult livers following a published protocol for organoids derivation and culture (Broutier et al., Nat Protoc. 2016;11:1724–1743). At passage 5, we isolated total RNA and performed bulk RNA-sequencing on 3 biological replicates from each region and genotype. Organoids were grown in Matrigel domes. At passage 5, RNA was collected using RNeasy Plus micro kit (Qiagen, 74034), sequencing library was prepared using TruSeq RNA SMP prep Kit-v2 SetA from Illumina (RS-122-2001) and it was sequenced on Illumina NextSeq 550 platform.

为研究阿拉基尔综合征（Alagille syndrome）小鼠模型中野生型与再生胆管的分子差异（Andersson、Chivukula、Hankeova等，《胃肠病学（Gastroenterology）》，2018；154(4)：1080-1095），我们参考已发表的类器官构建与培养方案（Broutier等，《自然·实验方案（Nature Protocols）》，2016；11：1724–1743），从成年肝脏的肝门区（pICOs）与外周区（pICOs）分离得到肝内胆管上皮类器官（intrahepatic cholangiocyte organoids）。在第5代传代时，我们提取总RNA，并对每个区域、每种基因型的3份生物学重复样本开展批量RNA测序（bulk RNA-sequencing）。类器官于基质胶（Matrigel）构建的穹顶状培养体系中培养。同样在第5代传代时，我们使用RNeasy Plus微型试剂盒（RNeasy Plus micro kit，Qiagen，74034）提取总RNA，采用Illumina的TruSeq RNA SMP制备试剂盒-v2 SetA（TruSeq RNA SMP prep Kit-v2 SetA，RS-122-2001）构建测序文库，并在Illumina NextSeq 550平台上完成测序。