MIR sequences recruit zinc finger protein ZNF768 to expressed genes

U2OS osteosarcoma cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco) and Raji B-cells in RPMI 1640 medium (Gibco) supplemented with 10% fetal calf serum (FCS, Bio&Sell), 2 mM L-glutamine (Gibco), 100 U/mL penicillin (Gibco), and 100 µg/mL streptomycin (Gibco) at 37°C at 8% or 5% CO2, respectively. Chromatin immunoprecipitation for ChIP-seq: Cells were crosslinked using a formaldehyde containing solution (10mM NaCl, 0,1mM EDTA pH 8, 0,05mM EGTA pH 8, 5mM HEPES pH 7.8 and 1% formaldehyde) for 10 minutes at 20°C, the reaction was quenched by the addition of glycin to a final concentration of 250uM for 5 minutes. Crosslinked cells were collected and washed twice with PBS before snap freezing in liquid nitrogen and storage at -80°C until subsequent use. Prior to sonication, the crosslinked cells were resuspended in lysis buffer (50mM Hepes pH 7.5, 140mM NaCl, 1mM EDTA pH 8, 10% glycerol, 0.75% NP-40, 0.25% Triton X-100, 1X protease inhibitor cocktail) at 4°C for 20 minutes. Nuclei were collected by centrifugation and washed in a second buffer (200mM NaCl, 1mM EDTA pH 8, 0.5mM EGTA pH 8, 10mM Tris pH 8, 1X protease inhibitor cocktail) for 10 minutes at 4°C then collected by centrifugation and resuspended in the shearing buffer (1mM EDTA pH 8, 0.5mM EGTA pH 8, 10mM Tris pH 8, 100mM NaCl, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine, 1X protease inhibitor cocktail). Sonication was carried out in a Bioruptor Pico ultrasounds water bath (Diagenode B01060001) for 30 cycles of 30 sec ON and 30 sec OFF pulses in 4°C water. Sonicated extracts were centrifuged at high speed in the presence of 0,1% of Triton X-100 and snap frozen in liquid nitrogen then stored at -80°C until subsequent use. Prior to ChIP, the ZNF768 mAb was coupled to protein-G coated magnetic beads (Dynabeads, life technologies) by incubation in 0,5% BSA PBS overnight at 4°C. Pre-coated beads were then washed and incubated with the sonicated chromatin extracts, the ChIP was carried out overnight at 4°C on a rotating wheel. The equivalent of 10 × 107 cells sonicated extract was used for each ZNF ChIP experiment for both cell lines. After incubation, the beads were washed 7 times with Wash buffer (50mM Hepes pH 7.6, 500mM LiCl, 1mM EDTA pH 8, 1% NP-40, 0.7% Na-Deoxycholate, 1X protease inhibitor cocktail) followed by one wash with TE-NaCl buffer (10mM Tris pH 8 and 1mM EDTA pH 8, 50mM NaCl). Immunoprecipitated chromatine was eluted by two sequential incubations with 100µl Elution buffer (50mM Tris pH 8, 10mM EDTA pH 8, 1% SDS) at 65°C for 15 minutes. The two eluates were pooled and incubated at 65°C for 12 hours to reverse-crosslink the chromatin followed by treatment with RNase A (0.2µg/mL) at 37°C for two hours and Proteinase K (0.2µg/mL) at 55°C for two hours. The DNA was isolated by phenol:chloroform: isoamylalcohol (25:24:1 pH 8) extranction followed by Qiaquick PCR Purification (Qiagen, Germany) and quantified with Qubit DS DNA HS Assay (ThermoFisher Scientific, USA). At least 1ng of ChIP DNA was used to prepare sequencing library with Illumina ChIP Sample Library Prep Kit (Illumina, USA) with a few optimizations to the protocol. The ChIP DNA was size selected using Ampure beads (Life technologies) to enrich for fragments < 400Bp prior to end-repair, 3’end adenylation and adapter ligation. Library fragments were then directly amplified by 10 cycles of PCR.

U2OS骨肉瘤细胞（U2OS osteosarcoma cells）培养于杜尔贝科改良伊格尔培养基（Dulbecco's modified Eagle's medium, DMEM，Gibco品牌）中；Raji B淋巴细胞则培养于RPMI 1640培养基（Gibco品牌），两种培养基均添加10%胎牛血清（fetal calf serum, FCS，Bio&Sell品牌）、2 mM L-谷氨酰胺（Gibco）、100 U/mL青霉素（Gibco）及100 μg/mL链霉素（Gibco），培养温度均为37℃，CO₂浓度分别为8%和5%。
用于染色质免疫沉淀测序（Chromatin immunoprecipitation sequencing, ChIP-seq）的染色质免疫沉淀实验流程如下：
首先对细胞进行交联：使用含甲醛的交联缓冲液（10 mM氯化钠、0.1 mM EDTA pH 8、0.05 mM EGTA pH 8、5 mM HEPES pH 7.8及1%甲醛）在20℃下孵育10分钟以交联细胞，随后加入终浓度为250 μM的甘氨酸终止交联反应，继续孵育5分钟。收集交联后的细胞，用磷酸盐缓冲液（PBS）洗涤两次，经液氮快速冷冻后于-80℃保存备用。
超声破碎前的细胞处理：将交联后的细胞重悬于裂解缓冲液（50 mM HEPES pH 7.5、140 mM氯化钠、1 mM EDTA pH 8、10%甘油、0.75% NP-40、0.25% Triton X-100及1×蛋白酶抑制剂混合物），4℃孵育20分钟。通过离心收集细胞核，使用第二洗涤缓冲液（200 mM氯化钠、1 mM EDTA pH 8、0.5 mM EGTA pH 8、10 mM Tris pH 8及1×蛋白酶抑制剂混合物）重悬并洗涤细胞核，4℃孵育10分钟后再次离心收集，随后将细胞核重悬于剪切缓冲液（1 mM EDTA pH 8、0.5 mM EGTA pH 8、10 mM Tris pH 8、100 mM氯化钠、0.1%脱氧胆酸钠、0.5% N-月桂酰肌氨酸钠及1×蛋白酶抑制剂混合物）。
超声破碎染色质：使用Bioruptor Pico超声波水浴仪（Diagenode B01060001），在4℃水浴中以30秒开启、30秒关闭的循环参数进行超声破碎，共30个周期。超声后的提取物添加0.1% Triton X-100后高速离心，经液氮快速冷冻后于-80℃保存备用。
染色质免疫沉淀（ChIP）操作：实验前，将ZNF768单克隆抗体（ZNF768 mAb）与包被蛋白G的磁珠（Dynabeads，Life Technologies品牌）在含0.5% BSA的PBS中4℃孵育过夜，完成抗体-磁珠偶联。将预偶联好的磁珠洗涤后，与超声破碎的染色质提取物混合，于4℃旋转孵育过夜以完成ChIP实验。两种细胞系的每次ZNF ChIP实验均使用相当于10×10⁷个细胞的超声染色质提取物。
孵育完成后，用洗涤缓冲液（50 mM HEPES pH 7.6、500 mM氯化锂、1 mM EDTA pH 8、1% NP-40、0.7%脱氧胆酸钠及1×蛋白酶抑制剂混合物）洗涤磁珠7次，再用TE-NaCl缓冲液（10 mM Tris pH 8、1 mM EDTA pH 8及50 mM氯化钠）洗涤1次。使用100 μL洗脱缓冲液（50 mM Tris pH 8、10 mM EDTA pH 8、1% SDS）经两次连续孵育，65℃下各孵育15分钟，洗脱免疫沉淀得到的染色质。合并两次洗脱液，65℃孵育12小时以逆转染色质交联，随后加入终浓度0.2 μg/mL的核糖核酸酶A（RNase A），37℃孵育2小时，再加入终浓度0.2 μg/mL的蛋白酶K（Proteinase K），55℃孵育2小时。
DNA纯化与定量：采用苯酚:氯仿:异戊醇（25:24:1，pH 8）萃取法分离DNA，随后经Qiaquick PCR纯化试剂盒（Qiagen，德国）纯化，使用Qubit dsDNA HS检测试剂盒（ThermoFisher Scientific，美国）对纯化后的DNA进行定量。
测序文库制备：取至少1 ng的ChIP DNA，使用Illumina ChIP样本建库试剂盒（Illumina，美国）并对标准建库流程进行少量优化以制备测序文库。在进行末端修复、3'端腺苷酸化及接头连接前，使用Ampure磁珠（Life Technologies）对ChIP DNA进行片段选择，富集长度小于400 bp的DNA片段。随后通过10轮PCR直接扩增得到的文库片段。