DRIP-qPCR validation

1. HEK293 cells were obtained from the National Collection of Authenticated Cell Culture of China (accession number: SCSP-5014) and cultured in DMEM supplemented with 10% FBS and penicillin/streptomycin (100U/ml) at 37°C with 5%CO2. Expression vector carrying NOTCH2NLC exon1 with different numbers of GGC repeats were prepared and transfected into HEK293 cells as described before (36). 2. For DRIP-qPCR, HEK293 cells (5×106) were washed in cold PBS, treated with 1ml PBS and collected by centrifuge at 600g for 5min at 4°C. Cells were treated with PK buffer (100mM NaCl, 10mM Tris pH 8.0, 1mM EDTA, 0.5% SDS), 6ul Proteinase K (300ug/ml) and 3ul Ribolock RNase inhibitor, followed by incubation at 37°C for 5h. DNA was extracted by phenol-chloroform-isoamyl alcohol in light phase lock tubes, precipitated with 1.5 ul glycogen, 1/10 volume sodium acetate (40 ul) and 2.5-fold volume of ethanol (1000 ul) at -80°C for 30 min, spin at 14000 rpm at 4°C for 15min, washed twice with 70% ethanol and re-suspend in 50 ul Tris elution buffer (10 mM Tris-HCl pH 8.0). 3. DNA was digested by 3ul HindIII (20,000units/ml, NEB), 3ul BsrGI (20,000units/ml, NEB), 3ul XbaI (20,000units/ml, NEB) and 3ul SspI (20,000units/ml, NEB). For R-loop validation, fragmented DNA was pretreated with 3ul RNase H (0297S, 10-unit total NEB) at 37°C. Digested DNA was purified by 200 ul TE buffer (10 mM Tris-Cl pH 8.0, 1 mM EDTA pH 8.0), extracted by phenol-chloroform-isoamyl alcohol, followed by ethanol precipitation. 4. Immunoprecipitations were performed by diluting 4ug of fragment DNA to 150ul 1× binding buffer (10 mM NaPO4 pH 7, 140 mM NaCl, 0.05% Triton X-100, 1× PI and Ribolock) and 0.4ug withdrawn to serve as input in qPCR. RNA-DNA hybrids were immunoprecipitated with 3ug of S9.6 overnight at 4°C. 5. Magnetic beads were washed 3 times with ChIP-dilution Buffer, incubated with Blocking buffer at RT for 2 hours on the rotating platform and washed 3 times with BSA+PBS. After removing BSA+PBS, DNA/antibody complex was added to beads and incubated for 2-3 hours at 4°C. Beads was washed three times in binding buffer (+0.3 x PI, 2 ul Ribolock) and elution was performed in 150 ul elution buffer (10 mM Tris pH 8, 1 mM EDTA, 1% SDS and 6 ul Proteinase K) for 45 min at 55°C. After adding 150ul TE buffer, DNA was extracted by phenol-chloroform-isoamyl alcohol, followed by ethanol precipitation. Eluted DRIP DNA was washed twice with ethanol, re-suspended in 50 ul H2O and analyzed by qPCR. Primers used for DRIP-qPCR were CAATGATACCGCGAGACCCA (AmpR-F), CTTGATCGTTGGGAACCGGA (AmpR-R), AAGGACGACGGCAACTACAA (EGFP-F) and CGATGTTGTGGCGGATCTTG (EGFP-R).

1. HEK293细胞（HEK293 cells）购自中国国家认证细胞库（National Collection of Authenticated Cell Culture of China），登录号（accession number）为SCSP-5014；于添加10%胎牛血清（Fetal Bovine Serum, FBS）及100U/ml青霉素/链霉素（penicillin/streptomycin）的DMEM培养基（DMEM）中，于37℃、5%CO₂培养箱内培养。携带不同GGC重复数的NOTCH2NLC外显子1（exon1）的表达载体，按照此前文献报道的方法（36）转染至HEK293细胞中。
2. 进行DRIP-qPCR实验时，取5×10⁶个HEK293细胞，用预冷的磷酸盐缓冲液（Phosphate Buffered Saline, PBS）洗涤后，加入1ml PBS重悬，于4℃、600g离心5min收集细胞。向细胞沉淀中加入PK缓冲液（100mM NaCl、10mM Tris pH 8.0、1mM EDTA、0.5% SDS）、6μl蛋白酶K（Proteinase K，300μg/ml）及3μl Ribolock核糖核酸酶抑制剂（Ribolock RNase inhibitor），37℃孵育5小时。采用酚-氯仿-异戊醇（phenol-chloroform-isoamyl alcohol）在锁相管（light phase lock tubes）中提取DNA，随后加入1.5μl糖原、1/10体积的乙酸钠（40μl）及2.5倍体积的乙醇（1000μl），于-80℃沉淀30min；4℃、14000rpm离心15min收集沉淀，用70%乙醇洗涤两次，最终重悬于50μl Tris洗脱缓冲液（10mM Tris-HCl pH 8.0）中。
3. 使用3μl HindIII（20000U/ml，NEB）、3μl BsrGI（20000U/ml，NEB）、3μl XbaI（20000U/ml，NEB）及3μl SspI（20000U/ml，NEB）对提取得到的DNA进行酶切。针对R环验证实验，将酶切后的DNA片段用3μl RNase H（0297S，总活性10单位，NEB）于37℃预处理。酶切产物用200μl TE缓冲液（10mM Tris-Cl pH 8.0、1mM EDTA pH 8.0）纯化，再经酚-氯仿-异戊醇抽提后进行乙醇沉淀。
4. 免疫沉淀实验操作如下：将4μg酶切后的DNA片段稀释至150μl 1×结合缓冲液（10mM NaPO4 pH 7、140mM NaCl、0.05% Triton X-100、1×蛋白酶抑制剂（Protease Inhibitor, PI）及Ribolock核糖核酸酶抑制剂），其中0.4μg留作qPCR的输入对照。使用3μg S9.6抗体，于4℃下过夜孵育以沉淀RNA-DNA杂交复合物。
5. 磁珠用ChIP稀释缓冲液（ChIP-dilution Buffer）洗涤3次，于室温下用封闭缓冲液（Blocking buffer）在旋转平台上封闭2小时，随后用含牛血清白蛋白的磷酸盐缓冲液（BSA+PBS）洗涤3次。弃去BSA-PBS后，将DNA-抗体复合物加入磁珠中，4℃孵育2-3小时。用含0.3×蛋白酶抑制剂及2μl Ribolock核糖核酸酶抑制剂的结合缓冲液洗涤磁珠三次，随后加入150μl洗脱缓冲液（10mM Tris pH 8、1mM EDTA、1% SDS及6μl蛋白酶K），55℃洗脱45min。向洗脱体系中加入150μl TE缓冲液后，用酚-氯仿-异戊醇抽提DNA，再经乙醇沉淀。洗脱得到的DRIP DNA用乙醇洗涤两次，重悬于50μl去离子水中，通过qPCR进行分析。DRIP-qPCR所用引物序列为：CAATGATACCGCGAGACCCA（AmpR-F）、CTTGATCGTTGGGAACCGGA（AmpR-R）、AAGGACGACGGCAACTACAA（EGFP-F）及CGATGTTGTGGCGGATCTTG（EGFP-R）。