人多能胚胎癌细胞中IGF2BP1靶基因的鉴定与验证方案。

Protocol for identification and validation of IGF2BP1 target genes in pluripotent human embryonic carcinoma cells.

作者信息

Myint Khine, Chuang Linda Shyue Huey, Matsuo Junichi, Ito Yoshiaki

机构信息

Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore.

出版信息

STAR Protoc. 2023 May 25;4(2):102338. doi: 10.1016/j.xpro.2023.102338.

DOI:10.1016/j.xpro.2023.102338

PMID:37243602

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10227443/

Abstract

We present a detailed protocol to identify and validate IGF2BP1 target genes in pluripotent human embryonic carcinoma cells (NTERA-2). We first identify the target genes through RNA-immunoprecipitation (RIP) sequencing. We then validate the identified targets through the use of RIP-qPCR assays, determine the mA status of target genes by mA-IP, and perform functional validation by quantifying changes in mRNA or protein expression levels upon knockdown of IGF2BP1 or methyltransferases in NTERA-2. For complete details on the use and execution of this protocol, please refer to Myint et al. (2022)..

摘要

我们展示了一个详细的方案，用于在多能性人类胚胎癌细胞（NTERA-2）中鉴定和验证IGF2BP1靶基因。我们首先通过RNA免疫沉淀（RIP）测序来鉴定靶基因。然后，我们通过RIP-qPCR分析来验证所鉴定的靶标，通过mA-IP确定靶基因的mA状态，并通过在NTERA-2中敲低IGF2BP1或甲基转移酶后定量mRNA或蛋白质表达水平的变化来进行功能验证。有关此方案的使用和执行的完整详细信息，请参考Myint等人（2022年）的文献。