ChIP-seq assay in human cancer cells

ChIP-seq assay was performed for the quantitative detection of the binding of TFs and histones upon acute stimulations. MCF7 cells were maintained at 37°C and 5% CO2 in Dulbecco's modified Eagle's medium (DMEM, GIBCO/Invitrogen), supplemented with 10% fetal bovine serum (FBS, GIBCO/Invitrogen). LNCAP cells were maintained at 37°C and 5% CO2 in Advanced RPMI 1640 medium (GIBCO/Invitrogen), supplemented with 10% fetal bovine serum (FBS, GIBCO/Invitrogen). For hormone treatments, cells were incubated at 37°C and 5% CO2 for at least 3 days in phenol red-free DMEM (GIBCO/Invitrogen) supplemented with 5% charcoal dextran-stripped FBS (GIBCO/Invitrogen). 17-β-Estradiol (E2; Steraloids, Inc.) was added to a final concentration of 100 nM. TNF⍺ (R&D systems) was added to a final concentration of 20 ng/ml. 5α-dihydrotestosterone (DHT, Sigma) was added to a final concentration of 100 nM. The ethanol (EtOH) vehicle control was 0.05% in all samples. For knock-down of Top1 genes, the siTop1_1 (SASI_Hs01_00047440, Sigma, CACAAAGAGAGGAAUGCUA[dT][dT], UAGCAAUCCUCUCUUUGUG[dT][dT]) and siTop1_3 (SASI_Hs02_00335354, Sigma, GACAAGAUCCGGAACCAGU[dT][dT], ACUGGUUCCGGAUCUUGUC[dT][dT]) were employed. For knock-down of Cbx3 genes, the siCbx3_1 (SASI_Hs01_00170890, Sigma, CCAAGAGGAUUUGCCAGAG[dT][dT], CUCUGGCAAAAUCCUCUUGG[dT][dT]) and siCbx3_2 (SASI_Hs01_00196532, Sigma, CCAAGAGGAUUUGCCAGAG[dT][dT], CUCUGGCAAAAUCCUCUUGG[dT][dT]) were employed. For ChIP-seq and PRO-seq experiments, cells were transfected in 10-cm plates in regular DMEM without antibiotics. 20 μl of 20 μM small interfering RNA (siRNAs) and Lipofectamine® 2000 reagent (Invitrogen Cat# 11668-019) were diluted in Opti-MEM I Reduced Serum Medium (Invitrogen Cat# 11058-021), and incubated for 6 h, and then changed to phenol red-free medium.

本研究采用染色质免疫共沉淀测序（ChIP-seq）实验技术，定量检测急性刺激下转录因子（Transcription Factors, TFs）与组蛋白的结合情况。MCF7细胞于37℃、5% CO₂条件下，在添加10%胎牛血清（Fetal Bovine Serum, FBS, GIBCO/Invitrogen）的达尔伯克改良伊格尔培养基（Dulbecco's modified Eagle's medium, DMEM, GIBCO/Invitrogen）中培养。LNCAP细胞于37℃、5% CO₂条件下，在添加10%胎牛血清（FBS, GIBCO/Invitrogen）的Advanced RPMI 1640培养基（GIBCO/Invitrogen）中培养。激素处理实验中，细胞于添加5%活性炭葡聚糖吸附胎牛血清（GIBCO/Invitrogen）的无酚红DMEM培养基（GIBCO/Invitrogen）中，于37℃、5% CO₂条件下孵育至少3天。向培养基中加入17β-雌二醇（17-β-Estradiol, E2; Steraloids公司），使其终浓度为100 nM。向培养基中加入肿瘤坏死因子α（TNFα, R&D Systems公司），使其终浓度为20 ng/ml。向培养基中加入5α-二氢睾酮（5α-dihydrotestosterone, DHT, Sigma公司），使其终浓度为100 nM。所有实验组均采用体积分数0.05%的乙醇（EtOH）作为溶剂对照。针对Top1基因的敲低实验，本研究使用了siTop1_1（货号SASI_Hs01_00047440，Sigma公司，序列为CACAAAGAGAGGAAUGCUA[dT][dT]、UAGCAAUCCUCUCUUUGUG[dT][dT]）与siTop1_3（货号SASI_Hs02_00335354，Sigma公司，序列为GACAAGAUCCGGAACCAGU[dT][dT]、ACUGGUUCCGGAUCUUGUC[dT][dT]）。针对Cbx3基因的敲低实验，本研究使用了siCbx3_1（货号SASI_Hs01_00170890，Sigma公司，序列为CCAAGAGGAUUUGCCAGAG[dT][dT]、CUCUGGCAAAAUCCUCUUGG[dT][dT]）与siCbx3_2（货号SASI_Hs01_00196532，Sigma公司，序列为CCAAGAGGAUUUGCCAGAG[dT][dT]、CUCUGGCAAAAUCCUCUUGG[dT][dT]）。针对ChIP-seq与PRO-seq实验，细胞于无抗生素的常规DMEM培养基中，在10 cm培养皿内进行转染。将20 μL 20 μM的小干扰RNA（small interfering RNA, siRNA）与Lipofectamine® 2000转染试剂（Invitrogen，货号11668-019）用Opti-MEM I 减血清培养基（Invitrogen，货号11058-021）稀释后孵育6小时，随后更换为无酚红培养基。