Real-time quantitative PCR analysis of Chromatin-associated snoRNA in human NB4 cells using Arraystar_384 platform

10 million cells were used for each sample. The cells were pre-treated with or without Doxorubicin (2µM for 4h) before collection and washed with ice-cold 1 × PBS. The nuclei pellet was further fractionated into nucleoplasmic fraction (supernatant) and chromatin pellet by using MWS buffer. All the buffers above were supplemented with protease inhibitor cocktail (Thermo Fisher Scientific) and Ribonuclease Inhibitors (Promega). The RNA precipitation solution (RPS) buffer was added into the cytoplasmic fraction and nucleoplasmic fraction immediately and each fraction was stored at -20°C for at least 1h. The two fractions were centrifuged and the resulting pellets were washed with 75% ethanol. All the pellets from the chromatin fraction, nucleoplasmic fraction and cytoplasmic fraction were added with 1mL TRIzol (Invitrogen) to extract RNA. The RNA pellet in TRIzol was fully dissolved by adding EDTA to the final concentration of 5mM and heat it to 65°C. The resulting RNAs from each fraction were subjected to DNase I (Sigma-Aldrich) treatment followed by phenol/chloroform extraction to remove any contaminant DNA. The RNA pellets of each fraction were then dissolved in equal volumes of RNase-free water and used for snoRNA qRT-PCR analyses. qPCR snoRNA expression profiling. NB4 cells were used and treated separately as indicated in the summary. Equal amount total RNA was used to analyze the gene expression.

每份样本使用1000万个细胞。收集细胞前，将细胞经或不经阿霉素（Doxorubicin，2µM浓度孵育4小时）预处理，随后用预冷的1×磷酸盐缓冲液（PBS）洗涤。使用MWS缓冲液将细胞核沉淀进一步分离为核质组分（上清液）与染色质沉淀组分。上述所有缓冲液均添加了蛋白酶抑制剂混合物（Protease inhibitor cocktail，赛默飞世尔科技，Thermo Fisher Scientific）与核糖核酸酶抑制剂（Ribonuclease Inhibitors，普洛麦格，Promega）。立即向细胞质组分与核质组分中加入RNA沉淀液（RPS）缓冲液，将各组分于-20℃下保存至少1小时。随后将两组分离心，所得沉淀用75%乙醇洗涤。将染色质组分、核质组分与细胞质组分的所有沉淀分别加入1mL TRIzol（Invitrogen）以提取RNA。向TRIzol中的RNA沉淀中加入乙二胺四乙酸（EDTA）至终浓度为5mM，并加热至65℃以充分溶解沉淀。将各组分所得RNA经脱氧核糖核酸酶I（DNase I，Sigma-Aldrich）处理后，通过酚/氯仿抽提去除残留污染DNA。将各组分的RNA沉淀用无RNase水以等体积溶解，用于小核RNA（snoRNA）定量实时聚合酶链反应（qRT-PCR）分析。小核RNA qPCR表达谱分析：本实验采用NB4细胞，按照实验概述中的方案分别进行处理，使用等量总RNA进行基因表达分析。