基因表达(CAGE)帽分析是一种对mRNA 5’ends进行全基因组定量映射的方法,以单核苷酸分辨率捕获RNA聚合酶II转录起始位点。本作品描述了一种低输入(SLIC-CAGE)协议,用于使用纳米图总RNA生成高质量库。
基因表达(CAGE)帽分析是一种用于RNA聚合酶II转录起始位点(TSSs)的单核苷酸分辨率检测方法。准确检测 TSS 可增强核心启动子的识别和发现。此外,可以通过双向转录启动的签名来检测有源增强剂。此处介绍用于执行超低输入载波 CAGE(SLIC-CAGE)的协议。CAGE协议的这种SLIC适应通过人工增加RNA量,通过使用在感兴趣的样品中加入的体外转录RNA载体混合物,从而从纳米图总量中制备,从而最大限度地减少了RNA损失RNA(即数千个细胞)。载体模拟预期的DNA库片段长度分布,从而消除由同质载体丰度引起的偏差。在协议的最后阶段,载体通过带宿主内分酶的降解被移除,目标库被放大。目标样本库受到保护,防止降解,因为宿主内分酶识别位点很长(在18至27bp之间),使得它们在真核基因组中存在的概率非常低。最终结果是DNA库为下一代测序做好准备。协议中的所有步骤,至顺序,可在 6 天内完成。承运人准备需要一个完整的工作日;然而,它可以大量制备,并保持在-80°C冷冻。一旦测序,读取可以处理获得全基因组的单核苷酸分辨率TSS.TSS可用于核心促进剂或增强剂发现,提供对基因调控的洞察。聚合到启动器后,数据还可用于 5′ 为中心的表达式分析。
基因表达(CAGE)的帽分析是一种用于单核苷酸解析全基因组图谱的RNA聚合酶II转录起始位点(TSSs)1。其定量性质还允许 5′ 端为中心的表达式分析。围绕TSS(约40 bp上游和下游)的区域是核心启动子,代表RNA聚合酶II和一般转录因子结合的物理位置(前2、3)。有关 TSS 的确切位置的信息可用于核心启动器发现和监测启动子动力学。此外,由于活性增强剂表现出双向转录的特征,CAGE数据也可用于增强剂发现和监测增强剂动力学4。CAGE方法最近越来越受欢迎,因为它在诸如ENCODE 5、modENCODE6和FANTOM项目7等备受瞩目的研究项目中有着广泛的应用和用途。此外,TSS 信息也被证明是重要的区分健康和疾病组织,因为疾病特异性的TSS可用于诊断目的8。
尽管有几种 TSS 映射方法可用(CAGE、RAMPAGE、STRT、纳米笼、纳米CAGE-XL、寡核封盖),但我们和其他人最近已经表明,CAGE 是最无偏见的方法来捕获假阳性数最少的真实 TSS9,10.最近的CAGE协议,nAnT-iCAGE11,是TSS分析最公正的协议,因为它避免使用限制性酶将片段切割成短标记,并且不使用PCR扩增。nAnT-iCAGE 协议的局限性是需要大量起始材料(例如,每个样品的总 RNA 量为 5 μg)。为了回答具体、与生物学相关的问题,通常无法获得如此大量的起始材料(例如,对于FACS排序的细胞或早期胚胎阶段)。最后,如果nAnT-iCAGE成功,每个样本只能获得1-2 ng的DNA库材料,从而限制了可实现的测序深度。
为了仅使用总RNA的纳米图进行TSS分析,我们最近开发了超低输入载波-CAGE 10(SLIC-CAGE,图1)。SLIC-CAGE 只需要总 RNA 的 10 纳克才能获得高复杂度库。我们的协议依赖于精心设计的合成RNA载体添加到感兴趣的RNA,实现总共5μg的RNA材料。合成载体在长度分布上模仿目标DNA库,以避免由过量的均质分子引起的潜在偏差。载体的序列基于大肠杆菌-tRNA合成酶基因(表1)的序列,原因有二。首先,最终库中载体的任何剩余,即使已测序,也不会映射到真核基因组。其次,由于大肠杆菌是一种嗜血菌物种,其内务管理基因针对适用于SLIC-CAGE的温度范围进行了优化。载体序列还嵌入了宿主内源内切酶识别位点,允许从载体RNA分子中提取的DNA进行特定降解。目标样本派生库保持不变,因为宿主内分酶识别站点很长(I-CeuI = 27 bp;I-SceI = 18 bp),在真核基因组中不太可能发现。在载体具体降解和通过大小排除去除碎片后,目标库被扩增,为下一代测序做好准备。根据起始RNA量(1-100纳克),预计在13-18PCR扩增周期之间。每个样本的最终DNA量在5-50 ng之间,产生足够的材料进行深度测序。当只使用总RNA的1-2纳克时,可以检测到真正的TSS;但是,这些库的复杂性预计会较低。最后,由于SLIC-CAGE基于nAnT-iCAGE协议11,因此在测序之前,它能对多达8个样本进行多路复用。
要成功进行 SLIC-CAGE 库准备,使用低结合提示和管来防止样品吸附导致样品损失至关重要。在涉及检索上清液的所有步骤中,建议恢复整个样本体积。由于该协议具有多个步骤,连续样本丢失将导致库失败。
如果 CAGE (nAnT-iCAGE) 没有正常执行,最好用相同总RNA样本的不同输入量(10 ng、20 ng、50 ng、100 ng、200 ng)测试SLIC-CAGE,并与使用5μg总RNA制备的nAnT-iCAGE库进行比较。如果 nAnT-iCAGE 库不成功(小于每个样本获得的 DNA 库的 0.5-1 ng),SLIC-CAGE 不太可能工作,并且需要将样本损失降至最低。
确保没有未封顶的降解RNA或rRNA的高质量库是关键步骤,第7节所述的封顶。非常重要的是,链球菌素珠子在洗涤缓冲液中彻底悬浮,在继续下一步洗涤步骤或 cDNA 洗脱之前,清除洗涤缓冲液。
如果第一轮载波降级后qPCR的结果显示适配器_f1和载波_f1引能的使用没有区别,仍建议继续使用该协议。如果在第二轮载波降级后,Ct 值的差异小于 5,则建议进行第三轮载波降级。我们从来没有发现第三轮降解是必要的,如果发生,建议取代宿主内分糖种群。
如果获得的库的最终量不足以进行测序,则可在协议中添加其他多轮 PCR 扩增。然后,PCR 扩增可以设置所需的最小扩增周期,以产生足够的材料进行测序,同时考虑到在尺寸选择中无法避免的样品损失。然后,应使用 SPRI 磁珠进行纯化或尺寸选择,直到去除所有小 (<200 bp) 片段(如果需要,使用 0.6:1 珠与采样比),并且库应使用 Picogreen 进行量化。
库可以在单端或成对端模式下排序。使用成对端测序,可以获得有关转录等形的信息。此外,由于使用随机引物(TCT-N 6,N6是随机六项机)进行逆转录,因此,来自测序3’端的信息可用作唯一分子标识符(UMI),以折叠PCR重复项。由于使用适量的PCR扩增周期(最多18个),以前发现使用UMIs是不必要的。
由于协议的核心依赖于nAnT-iCAGE1,SLIC-CAGE使用8个条形码。因此,当前不支持多路复用超过 8 个示例。此外,SLIC-CAGE 和 nAnT-iCAGE 都不适合捕获小于 200 bp 的 RNA,因为协议旨在通过使用 AMPure XP 磁珠排除尺寸来去除链接器和 PCR 人工制品。
SLIC-CAGE 是唯一一种使用总RNA材料的纳米图绘制转录起始位点的无偏低输入单核苷酸分辨率方法。替代方法依赖于逆转录酶的模板切换活性,以条形码封盖RNA,而不是捕获上限(例如,NanoCAGE15和 NanoPARE16)。由于模板切换,这些方法在TSS检测中表现出序列特定的偏差,导致误报TS的数量减少和真TSS9、10的数量减少。
The authors have nothing to disclose.
这项工作得到了威尔康信托赠款(106954)的支持,该赠款授予了B.L.和医学研究委员会(MRC)核心基金(MC-A652-5QA10)。N.C.得到EMBO长期研究金的支持(EMBO ALTF 1279-2016);E. P. 得到了英国医学研究理事会的支持;B. L. 得到了英国医学研究理事会(MC UP 1102/1)的支持。
2-propanol, Bioultra, for molecular biology, ≥99.5% | Sigma-Aldrich | 59304-100ML-F | Used in RNAclean XP purification. |
3' linkers | Sequences are described in Murata et al 2014 and Supplementary Table 1 of this manuscript. Annealing of strands to produce 3'linkers is described in the supplementary of this protocol. | ||
5' linkers | Sequences are described in Murata et al 2014 and Supplementary Table 1 of this manuscript. Annealing of strands to produce 5'linkers is described in the supplementary of this protocol. | ||
Agencourt AMPure XP, 60 mL | Beckman Coulter | A63881 | Purification of DNA |
Agencourt RNAClean XP Kit | Beckman Coulter | A63987 | Purification of RNA and RNA:cDNA hybrids in CAGE steps. |
Axygen 0.2 mL Polypropylene PCR Tube Strips and Domed Cap Strips | Axygen (available through Corning) | PCR-0208-CP-C | Or any 8-tube PCR strips (used only for water and mixes). |
Axygen 1 x 8 strip domed PCR caps | Axygen (available through Corning) | PCR-02CP-C | Caps for PCR plates. |
Axygen 1.5 mL Maxymum Recovery Snaplock Microcentrifuge Tube | Axygen (available through Corning) | MCT-150-L-C | Low-binding 1.5 ml tubes, used for enzyme mixes or sample concentration. |
Axygen 96 well no skirt PCR microplate | Axygen (available through Corning) | PCR-96-C | Low-binding PCR plates – have to be used for all steps in the protocol. Note that plates should be cut to contain 2 x 8 wells for easier visibility of the samples |
Bioanalyzer (or Tapestation): RNA nano and HS DNA kits | Agilent | To determine quality of RNA, efficient size selection and final quality of the library (Tapestation can also be used) | |
Biotin (Long Arm) Hydrazide | Vector laboratories | SP-1100 | Biotinylation/tagging |
Cutsmart buffer | NEB | Restriction enzyme buffer | |
Deep Vent (exo-) DNA Polymerase | NEB | M0259S | Second strand synthesis |
DNA Ligation Kit, Mighty Mix | Takara | 6023 | Used for 5' and 3'-linker ligation |
dNTP mix (10 mM each) | ThermoFisher Scientific | 18427013 | dNTP mix for production of carrier templates (or any dNTPs suitable for PCR) |
Dynabeads M-270 Streptavidin | Invitrogen | 65305 | Cap-trapping. Do not use other beads as these are optimised with the buffers used. |
DynaMag-2 Magnet | ThermoFisher Scientific | 12321D | Magnetic stand for 1.5 ml tubes – used to prepare Streptavidin beads. |
DynaMag-96 Side Skirted Magnet | ThermoFisher Scientific | 12027 | Magnetic stand for PCR plates (96 well-plates) – used with cut plates to contain 2 x 8 wells. |
Ethanol, BioUltra, for molecular biology, ≥99.8% | Sigma-Aldrich | 51976-500ML-F | Used in AMPure washes. Any molecular biology suitable ethanol can be used. |
Exonuclease I (E. coli) | NEB | M0293S | Leftover primer degradation |
Gel Loading Dye, Purple (6x), no SDS | NEB | B7025S | agarose gel loading dye |
HiScribe T7 High Yield RNA Synthesis Kit | New England Biolabs | E2040S | Kit for carrier in vitro transcription |
Horizontal electrophoresis apparatus | purification of carrier DNA templates from agarose gels | ||
I-Ceu | NEB | R0699S | Homing endonuclease used for carrier degradation. |
I-SceI | NEB | R0694S | Homing endonuclease used for carrier degradation. |
KAPA HiFi HS ReadyMix (2x) | Kapa Biosystems (Supplied by Roche) | KK2601 | PCR mix for target library amplification |
KAPA SYBR FAST qPCR kit (Universal) 2x | Kapa Biosystems (Supplied by Roche) | KK4600 | qPCR mix to assess degradation efficiency and requiered number of PCR amplification cycles |
Micropipettes and multichannel micropipettes (0.1-10 µl, 1-20 µl, 20-200 µ) | Gilson | Use of Gilson with the low-binding Sorenson tips is recommended. Other micropippetes might not be compatible.. Different brand low-binding tips may not be of equal quality and may increase sample loss. | |
Microplate reader | For Picogreen concentration measurement of the final library. Microplates are used to allow small volume measurement and reduce sample waste. | ||
nuclease free water | ThermoFisher Scientific | AM9937 | Or any nuclease (DNase and RNase) free water |
PCR thermal cycler | incubation steps and PCR amplficication | ||
Phusion High-Fidelity DNA Polymerase | ThermoFisher Scientific | F530S | DNA polymerase for amplification of carrier templates (or any high fidelity polymerase) |
QIAquick Gel Extraction Kit (50) | Qiagen | 28704 | Purification of carrier PCR templates from agarose gels. |
qPCR machine | determining PCR amplification cyle number and degree of carrier degradation | ||
Quant-iT PicoGreen dsDNA Reagent | ThermoFisher Scientific | P11495 | Used to measure final library concentration – recommended as, in our hands, it is more accurate and reproducible than Qubit. |
Quick-Load Purple 100 bp DNA Ladder | NEB | N0551S | DNA ladder |
Quick-Load Purple 1 kb Plus DNA Ladder | NEB | N0550S | DNA ladder |
Ribonuclease H | Takara | 2150A | Digestion of RNA after cap-trapping. |
RNase ONE Ribonuclease | Promega | M4261 | Degradation of single stranded RNA not protected by cDNA. |
RNase-Free DNase Set | Qiagen | 79254 | Removal of carrier DNA templates after in vitro transcription. |
RNeasy Mini Kit | Qiagen | 74104 | For cleanup of carrier RNA from in vitro transcription or capping |
Sodium acetate, 1 M, aq.soln, pH 4.5 RNAse free | VWR | AAJ63669-AK | Or any nuclease (DNase and RNase) free solution |
Sodium acetate, 1 M, aq.soln, pH 6.0 RNAse free | Or any nuclease (DNase and RNase) free solution | ||
Sodium periodate | Sigma-Aldrich | 311448-100G | Oxidation of vicinal diols |
Sorenson low binding aerosol barrier tips, MicroReach Guard, volume range 10 μL, Graduated | Sorenson (available through SIGMA-ALDRICH) | Z719390-960EA | Low-binding tips – recommended use throughout the protocol to minimise sample loss. |
Sorenson low binding aerosol barrier tips, MultiGuard, volume range 1000 μL , Graduated | Sorenson (available through SIGMA-ALDRICH) | Z719463-1000EA | Low-binding tips – recommended use throughout the protocol to minimise sample loss. |
Sorenson low binding aerosol barrier tips, MultiGuard, volume range 20 μL , Graduated | Sorenson (available through SIGMA-ALDRICH) | Z719412-960EA | Low-binding tips – recommended use throughout the protocol to minimise sample loss. |
Sorenson low binding aerosol barrier tips, MultiGuard, volume range 200 μL , Graduated | Sorenson (available through SIGMA-ALDRICH) | Z719447-960EA | Low-binding tips – recommended use throughout the protocol to minimise sample loss. |
SpeedVac Vacuum Concentrator | concentrating samples in various steps to lower volume | ||
SuperScript III Reverse Transcriptase | ThermoFisher Scientific | 18080044 | Used for reverse transcription (1st CAGE step) |
Trehalose/sorbitol solution | Preparation is described in Murata et al 2014. | ||
Tris-HCl, 1M aq.soln, pH 8.5 | 1 M solution, DNase and RNase free | ||
tRNA (20 mg/mL) | tRNA solution. Preparation is described in Murata et al 2014. | ||
UltraPure Low Melting Point Agarose | ThermoFisher Scientific | 16520050 | Or any suitable pure low-melt agarose. |
USB Shrimp Alkaline Phosphatase (SAP) | Applied Biosystems (Provided by ThermoFisher Scientific) | 78390500UN | |
USER Enzyme | NEB | M5505S | Degradation of 3'linker's upper strand, Uracil Specific Excision Reagent/Enzyme |
Vaccinia Capping System | NEB | M2080S | Enzymatic kit for in vitro capping of carrier molecules |
Wash buffer A | Cap trapping washes. Preparation is described in Murata et al 2014. | ||
Wash buffer B | Cap trapping washes. Preparation is described in Murata et al 2014. | ||
Wash buffer C | Cap trapping washes. Preparation is described in Murata et al 2014. |