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DNA to 150ul 1× binding buffer (10 mM NaPO4 pH 7, 140 mM NaCl, 0.05% Triton X-100, 1× PI and Ribolock) and 0.4ug withdrawn to serve as input in qPCR.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细胞购自中国国家认证细胞库（National Collection of Authenticated Cell Culture of China，登录号：SCSP-5014），于添加10%胎牛血清（FBS）和100U/ml青霉素/链霉素的达尔伯克改良伊格尔培养基（DMEM）中，在37℃、5%CO₂条件下培养。按照此前报道的方法（参考文献36），制备携带不同GGC重复数的NOTCH2NLC外显子1的表达载体，并转染至HEK293细胞中。
2. 针对DNA-RNA免疫沉淀定量PCR（DRIP-qPCR）实验，取5×10⁶个HEK293细胞，用预冷的磷酸盐缓冲液（PBS）洗涤，加入1ml PBS后于4℃、600g离心5min收集细胞。随后用PK缓冲液（100mM NaCl、10mM Tris pH 8.0、1mM EDTA、0.5% SDS）、6μl蛋白酶K（300μg/ml）及3μl Ribolock核糖核酸酶抑制剂处理细胞，于37℃孵育5h。采用酚-氯仿-异戊醇在锁相管中提取DNA，加入1.5μl糖原、1/10体积（40μl）乙酸钠及2.5倍体积（1000μl）乙醇，于-80℃静置30min沉淀DNA，4℃下14000rpm离心15min，用70%乙醇洗涤沉淀两次，最后用50μl Tris洗脱缓冲液（10mM Tris-HCl pH 8.0）重悬DNA。
3. 用3μl HindIII（20000U/ml，NEB）、3μl BsrGI（20000U/ml，NEB）、3μl XbaI（20000U/ml，NEB）及3μl SspI（20000U/ml，NEB）对DNA进行酶切。为验证R环，将酶切后的DNA片段用3μl核糖核酸酶H（RNase H，0297S，总活性10U，NEB）于37℃预处理。酶切后的DNA用200μl TE缓冲液（10mM Tris-Cl pH 8.0、1mM EDTA pH 8.0）纯化，再经酚-氯仿-异戊醇提取后进行乙醇沉淀。
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（PI）及Ribolock），并取0.4μg作为定量PCR（qPCR）的输入对照。将RNA-DNA杂交体与3μg S9.6抗体于4℃孵育过夜进行免疫沉淀。
5. 磁珠用ChIP稀释缓冲液洗涤3次，于室温下在旋转平台上用封闭缓冲液孵育2h，再用牛血清白蛋白-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，于4℃孵育2~3h。用含0.3×PI及2μl Ribolock的结合缓冲液洗涤磁珠3次，随后在150μl洗脱缓冲液（10mM Tris pH 8、1mM EDTA、1% SDS及6μl蛋白酶K）中于55℃孵育45min进行洗脱。加入150μl TE缓冲液后，用酚-氯仿-异戊醇提取DNA并进行乙醇沉淀。洗脱得到的DRIP DNA用乙醇洗涤两次，重悬于50μl无菌水中，随后通过qPCR进行分析。本实验所用引物为CAATGATACCGCGAGACCCA（AmpR-F）、CTTGATCGTTGGGAACCGGA（AmpR-R）、AAGGACGACGGCAACTACAA（EGFP-F）及CGATGTTGTGGCGGATCTTG（EGFP-R）。