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| { "title": "RIP-seq Protocol", "steps": [ { "title": "1. cell cuture", "substeps": [ { "title": "1.1", "content": "NCCIT细胞每个样品养到6孔板的两个孔长满,大概5~6百万细胞", "image": "https://fanyiming.life/images/avatar.jpg" } ] }, { "title": "2. Beads preparation", "substeps": [ { "title": "2.1", "content": "每个样品用40μl ProteinG Dyna beads,取样品数 * 40μL", "image": "" }, { "title": "2.2", "content": "加0.9 mL Lysis Buffer (加1% protease inhibitor cocktail III)洗四次", "image": "" }, { "title": "2.3", "content": "用200μL Lysis Buffer (加1% protease inhibitor cocktail III)重悬,每40 μL beads添加5 μg抗体", "image": "" } ] }, { "title": "3. UV cross-linking", "substeps": [ { "title": "3.1", "content": "从培养箱中取出细胞,弃去培养基,加0.5 mL 预冷的PBS (这里的PBS可以不加protease inhibitor)", "image": "" }, { "title": "3.2", "content": "吸弃PBS,敞开盖子,细胞裸露在空气中,放入UV交联仪", "image": "" }, { "title": "3.3", "content": "400 mJ/cm 交联。(对于本实验室仪器,点energy,输入4000,然后Start)", "image": "" }, { "title": "3.4", "content": "每个孔加入1mL PBS(+protease inhibitor),对于NCCIT来说,直接用枪头吹打,收集到2mL EP管中。", "image": "" }, { "title": "3.5", "content": "台盼蓝法对细胞进行计数,记细胞总数,死活都要。", "image": "" }, { "title": "3.6", "content": "每个样品取用5M细胞,4℃,5000rpm离心5min收集", "image": "" } ] }, {
"title": "4 细胞裂解和免疫沉淀", "substeps": [ { "title": "4.1", "content": "离心结束后,弃上清,每个管子加入150 μL lysis buffer (+cocktail),转移到1.5 mL lowBind EP管中。(因为下一步要震荡,我们实验室的那个机器只能用1.5mL的管子),冰上放置5min。", "image": "" }, { "title": "4.2", "content": "每管加入14 μL Turbo DNase (Ambion, AM2238)和7.5 μL RNase Inhibitor,37℃下1100rpm震荡5min.", "image": "" }, { "title": "4.3", "content": "4℃, 14000g离心10min,小心取出,保持沉淀在底部。", "image": "" }, { "title": "4.4", "content": "准备新的EP管,取出2~3μL作为Input,放到-20℃暂存。", "image": "" }, { "title": "4.5", "content": "把包好抗体的beads分成每20μL最初的beads体积一个1.5mL EP管,磁力架放置1min,弃上清。", "image": "" }, { "title": "4.6", "content": "每个含有beads的EP管中加入75μL lysates (每个样品2个replicates),4℃慢速旋转孵育2h~4h。", "image": "" }, { "title": "4.7", "content": "用High Stringent Buffer(+cocktail)洗beads,一次,4℃慢速旋转10min。", "image": "" }, { "title": "4.8", "content": "用High Salt Buffer(+cocktail)洗beads,一次,4℃慢速旋转10min。", "image": "" }, { "title": "4.9", "content": "用Low Salt Buffer(+cocktail)洗beads,一次,4℃慢速旋转10min。", "image": "" }, { "title": "4.10", "content": "把Input从冰箱中取出,跟刚才洗好的replicate1和replicate2一起,用50 μL Proteinase K digestion Buffer (+ 2.5μL RNase Inhibitor和5μL proteinase K)重悬beads,50℃, 1100rpm振荡 2h。\n (beads就呆在管子里,之后RNAC抽提的时候会跑到phase-lock tubes的底部。)", "image": "" } ] }, { "title": "5 RNA抽提", "substeps": [ { "title": "5.1", "content": "准备Phase-Lock tubes, 室温16000g离心30s。", "image": "" }, { "title": "5.2", "content": "把样品取出,加150μl lysis Buffer(+cocktail)补全体积到200μL。", "image": "" }, { "title": "5.3", "content": "加入200 μL RNA抽提液(25:24:1),剧烈震荡50次,室温13200rpm离心5min。", "image": "" }, { "title": "5.4", "content": "加入200μL氯仿,剧烈震荡50次,室温13200rpm离心5min。", "image": "" }, { "title": "5.5", "content": "上清液转移到新的1.5mL LowBind EP管中。", "image": "" } ] }, { "title": "6 DNA消化后再次抽提", "substeps": [ { "title": "6.1", "content": "每管加入18μL Turbo DNase,37℃, 1100rpm振荡15min。", "image": "" }, { "title": "6.2", "content": "准备Phase-Lock tubes, 室温16000g离心30s。", "image": "" }, { "title": "6.3", "content": "反应结束后,转移全部溶液(大约200μL)至Phase-Lock tubes中。", "image": "" }, { "title": "6.4", "content": "加入200 μL RNA抽提液(25:24:1),剧烈震荡50次,室温13200rpm离心5min。", "image": "" }, { "title": "6.5", "content": "加入200μL氯仿,剧烈震荡50次,室温13200rpm离心5min。", "image": "" }, { "title": "6.6", "content": "上清液转移到新的1.5mL LowBind EP管中。", "image": "" } ] }, { "title": "7 核酸醇沉", "substeps": [ { "title": "7.1", "content": "加入20μL 3M NaAc和1μL Glycogen (20μg/μL),混匀。", "image": "" }, { "title": "7.2", "content": "加入600 μL预冷的无水乙醇。", "image": "" }, { "title": "7.3", "content": "混匀后,放-80℃醇沉(大于2h,长则一星期)。", "image": "" }, { "title": "7.4", "content": "4℃, 14000g离心20min。", "image": "" }, { "title": "7.5", "content": "弃上清,加500μl预冷的75%乙醇。4摄氏度,14000rpm,离心5min。", "image": "" }, { "title": "7.6", "content": "弃上清,4摄氏度,14000rpm,离心1min,用10μl枪头吸干上清。", "image": "" }, { "title": "7.7", "content": "风干3min,加适量体积的Nuclease-Free Water,溶解。(对于本RIP-seq来说加12μL洗脱)", "image": "" }, { "title": "7.8", "content": "用NanoDrop测浓度", "image": "" } ] }, { "title": "8 二代测序建库", "substeps": [ { "title": "8.1打断", "content": "按浓度最低的取3μL至八联排,其他取跟它相同的ng数,用RNase-Free Water补全至3μL。每管加入3μL Frag/Elute Buffer。94℃打断8min,4℃ hold", "image": "" }, { "title": "8.2一链合成", "content": "每管加入4.8μL RT strand specificity Reagent + 1.2μL First Strand synthesis enzyme mix,可先预混。\n 25℃,10min → 42℃,15min → 70℃,15min → 4℃,hold", "image": "" }, { "title": "8.3二链合成", "content": "每管加入22μL RNase-Free Water + 4μL second strand synthesis reaction buffer with dUTP + 2μL Second strand synthesis enzyme mix,可先预混,总体积40μL。16℃孵育1h", "image": "" }, { "title": "8.4用beads纯化", "content": "每管加入72μL NFTmag NGS DNA clean Beads,混匀,室温静置5min,磁力架静置5min,弃上清。\n将tube保持在磁力架上,加入100μL 80%乙醇,放30s,弃上清。再加入100μL 80%乙醇,放30s,弃上清。室温干燥3min,加入19.5μL low-EDTA TE,吹打混匀,室温静置2min,磁力架1min,吸取18.5μL上清至另一八联排中", "image": "" }, { "title": "8.5末端修复", "content": "上一步的Tube中,每管加入5μL End-Prep Buffer和1.5μL End-Prep enzymes(总体积25μL)。20℃,30min → 65℃,30min → 4℃,hold", "image": "" }, { "title": "8.6接头连接", "content": "上一步的Tube中,每管加入8.25μL Ligation buffer,1.5μL Ligase Mix和1.25μL Truncated Adapter。其中,Truncated Adapter不要跟另外二者预混,最后单独加,以免形成接头二聚体。22℃反应15min(总体积36μL,热盖关闭)", "image": "" }, { "title": "8.7片段分选", "content": "上一步的Tube中,加入14μL Nuclease-Free Water,成50μL体系。加15μL beads,混匀,室温静置5min,磁力架5min。转移上清至另一八联排中(!!不要丢掉上清!!),加10μL beads混匀,室温静置5min,磁力架5min,弃上清。100μL 80%乙醇洗两次。风干3min,加10.5 μL low-EDTA TE,吹打混匀,室温静置2min,磁力架1min,吸取10 μL上清至另一八联排中", "image": "" }, { "title": "8.8PCR文库扩增", "content": "取新的八联排,每管加入6.25μL 2XPCR mix和0.125μL UDG enzyme(可以看出后者用量非常小,二者先预混)。然后分别加入4.875μL上一步得到的“片段分选产物”,再各自加入不同的Dual-index primer 1.25 μL,在实验记录本上记清楚加了哪几个primer,分别对应哪个样品。37℃,10min → 98℃,1min → 98℃,10s → 60℃,15s → 72℃,30s (goto step3, 8X) → 72℃,1min → 4℃,hold。返回去8次,即9个cycles", "image": "" }, { "title": "8.9跑胶检测扩增效果", "content": "提前配置1%琼脂糖凝胶,取2μL PCR产物跑胶。根据结果,看是否有个别样品需要多加1~2个循环。加完心里有数的话就不用再跑胶了。", "image": "" }, { "title": "8.10 PCR产物回收", "content": "按体积加1:1的beads,混匀,室温5min,磁力架5min,弃上清,100μL 80%乙醇洗两次,风干2min,加15μL low-EDTA TE混匀,室温2min,磁力架1min,吸取上清至新的八联排。", "image": "" }, { "title": "8.11 Qubit核酸定量", "content": "每个Qubit管中加入199μL Qubit反应液体,加入1μL样品,混匀。放入仪器检测,读数,记录", "image": "" }, { "title": "8.12混样", "content": "最低浓度的样品取一半(保证有一次失误补救的机会),其他取相同ng数(至少大于5ng)。可以几个一组混样(而不是全部混成1个样)方便加测。要注意记录每个样品取了几微升,然后按照总体积的80%加入beads,混匀,室温5min,磁力架5min,弃上清,100μL 80%乙醇洗两次,风干2min,加20μL Low-EDTA TE洗脱(如果样品数少,减少洗脱体积)", "image": "" }, { "title": "8.13再次Qubit定量", "content": "因为填测序订单需要写浓度,所以需要再测一遍Qubit,199μL Qubit反应液体,加入1μL样品,混匀。放入仪器检测,读数,记录", "image": "" }, { "title": "8.14填写订单,送测", "content": "麻烦易出错的地方在于填好每个子样品对应的Dual-Index Primer,错了不只耽误自己同时还耽误一起上机的其他人。", "image": "" } ] } ] }
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