RNA Immunoprecipitation in macrophages using NLRC4 and Caspase11 and high-throughput sequencing of isolated lncRNA

At the epigenetic level, lncRNAs play important roles in cell differentiation and function. We identified novel long-stranded non-coding RNA (GM1082) in BMDMs, which may play an important role in macrophage differentiation and function.To confirm the interaction between LNCRNA and NLRC4 and Caspase11, we performed RNA immunoprecipitation using NLRC4 and Caspase11 antibodies, and high-throughput sequencing of the obtained RNA. Mouse bone marrow cells were induced in vitro by adding 20ng/ml of M-csf in DMEM medium.After 6 days of M-CSF induction, BMDMs were exposed to flagellin.Cell lysates were incubated with normal rabbit IgG, NLRC4 and caspase11 antibody.RNAs were isolated from the Protein–RNA complexes with phenol-chloroform and subjected to library construction.

在表观遗传水平，长链非编码RNA（long non-coding RNAs, lncRNAs）在细胞分化与功能调控中发挥重要作用。本研究在骨髓源性巨噬细胞（bone marrow-derived macrophages, BMDMs）中鉴定出一种新型长链非编码RNA（GM1082），其可能在巨噬细胞分化及功能行使中具有关键作用。为验证该长链非编码RNA与NLRC4及半胱天冬氨酸蛋白酶11（Caspase-11）的相互作用，我们采用NLRC4与Caspase-11抗体开展RNA免疫沉淀实验，并对富集得到的RNA进行高通量测序。实验中，我们在DMEM培养基中添加20ng/ml的巨噬细胞集落刺激因子（Macrophage Colony-Stimulating Factor, M-CSF），体外诱导小鼠骨髓细胞分化。经M-CSF诱导6天后，用鞭毛蛋白刺激骨髓源性巨噬细胞。将细胞裂解液分别与正常兔免疫球蛋白G（normal rabbit IgG）、NLRC4抗体及Caspase-11抗体共孵育。通过酚-氯仿法从蛋白质-RNA复合物中分离RNA，并完成文库构建。