WO2023028954A1 - 高通量单细胞靶向测序的试剂和方法 - Google Patents
高通量单细胞靶向测序的试剂和方法 Download PDFInfo
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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Definitions
- the present application generally relates to the field of molecular biology, and specifically provides methods, components, kits and systems including for high-throughput single-cell target sequencing.
- Single-cell transcriptome sequencing is the most popular technology in the field of biology in recent years. Its ultra-high resolution can realize accurate analysis of sample information, and has great application potential in many fields of biology. For example, studying the important influence of tumor heterogeneity on disease development and drug intervention is used to dissect the diversity of immune receptors and translate into vaccination, cancer and autoimmune diseases.
- RNA splicing events [1,2]
- single-cell transcriptome sequencing is limited by the current short-read mRNA library sequencing technology, there is obvious selectivity for the 3' end or 5' end of the transcript, and it cannot effectively capture gene sequences far from the 3' end or 5' end, making it more difficult to detect complex gene rearrangements and RNA splicing .
- each newly differentiated T or B lymphocyte in the immune system carries a different antigen receptor, and DNA rearrangement changes 450 nucleotides at the 5' end of T cell or B cell antigen receptor mRNA [3] .
- B lymphocytes they use additional DNA rearrangements to "isotype switch" between nine alternative constant region sequences comprising 1000-1500 nucleotides at the 3' end of the heavy chain mRNA [4] and use alternative mRNA splicing changes the nucleotides at the 3' end of IGH mRNA to secrete the encoded receptor as an antibody [5] .
- complex gene rearrangements and alternative splicing events generate pathological cellular diversity in cancer cells. Therefore, methods that can capture these sequence changes that occur across the entire length of an mRNA molecule at single-cell resolution and combine this information with gene expression signatures are urgently needed.
- the present application provides methods, compositions, kits and systems for high-throughput single-cell target sequencing.
- the present application provides a method for analyzing cellular gene expression levels and target gene sequences at the single-cell level, wherein the method includes:
- each barcode oligonucleotide of the plurality of barcode oligonucleotides includes a cell barcode and a unique molecular identifier ( UMI), wherein the first barcode oligonucleotide of the plurality of barcode oligonucleotides each comprises a polyT sequence capable of binding a polyA tail of a first messenger ribonucleic acid (mRNA) target;
- mRNA messenger ribonucleic acid
- Circular cDNA is used for enrichment of target genes.
- the plurality of barcode oligonucleotide sequences cover two different sequencing primer binding sites; wherein, the binding sites can be used as PCR primer binding sequences to amplify cDNA sequences.
- the multiple barcode oligonucleotide sequences cover two different sequencing primer binding sites; wherein, the binding sites can be used as sequencing primer binding sites for library sequencing.
- said cDNA circularization is performed by an enzymatic reaction.
- the cDNA circularizing enzyme is DNA ligase.
- the cDNA circularizing enzyme is DNA polymerase.
- the enrichment of the target gene is carried out by means of inverse PCR using circular cDNA as a template.
- the way of obtaining the information is gene sequencing.
- the method of the matching analysis is performed by pairing after Cell barcode identification.
- said nucleic acid target comprises ribonucleic acid (RNA), messenger RNA (mRNA) and deoxyribonucleic acid (DNA), and/or wherein said nucleic acid target comprises a cell from, in a cell and/or on a cell surface nucleic acid target.
- RNA ribonucleic acid
- mRNA messenger RNA
- DNA deoxyribonucleic acid
- the source of the enriched target gene can be any vertebrate.
- Figure 1 is a schematic diagram of full-length transcript enrichment
- Figure 2 is a schematic diagram of cDNA circularization and full-length TCR enrichment
- Fig. 3 is the mass detection peak diagram of the product of full-length transcript amplification
- Fig. 4 is the mass detection peak diagram of the product of full-length TCR amplification
- Figure 5 is the TCR TOP10 clonotype
- FIG. 6 shows the quality control results of TCR library sequencing.
- Single-cell transcriptome sequencing combined with single-cell targeted sequencing technology can simultaneously analyze the cell type and target gene sequence information of the transcriptome, including cell mutation information, and is a powerful tool for studying the relationship between tumor cell development, targeted drugs, and gene hotspot mutations. tool.
- Single-cell transcriptome combined with targeted mutation can accurately identify the mutated cell type and provide reference for clinical medicine. At the same time, it can dynamically monitor the changes in the type and frequency of mutations during the course of medication. This technology is to circularize a part of the double-stranded cDNA and design strand-specific primers to enrich the target gene far from the 3' end by reverse PCR technology.
- the single-cell transcriptome can also obtain the target gene of interest and immune receptor sequence.
- the method disclosed in this paper can not only obtain high-quality single-cell transcriptome data, but also obtain interesting hotspot mutation information at a much lower sequencing depth than transcriptome according to customer needs.
- the technology has the following characteristics: (1) High-throughput: it can detect mutations in thousands of cells at the same time; (2) Deep customization: corresponding enrichment primers can be designed according to different needs of customers; (3) Cost-effective : Experimental procedures are highly compatible with single-cell transcriptome workflows. It only needs to customize the primer sequence and construct the corresponding enrichment library to achieve the capture of the target region.
- the Oligo sequence of Barcoding Beads contains illumina Read1 sequencing primer sequence, cell label (Cell Barcode), molecular label (UMI) and PolyT nucleotide sequence.
- the enrichment of the full-length transcriptome was amplified through the PCR handle sequence at the 5' end of the Barcoding Beads (adapted to the sequencing primers of the illumina next-generation sequencing platform) and the TSO sequence added during the reverse transcription process.
- the obtained cDNA was first circularized, and then PCR amplification was performed using the Universal sequence on Barcoding Beads and TCR-specific primers (C region sequence) to complete the enrichment of full-length TCR. Take 50ng of full-length TCR-enriched products for library construction.
- wash Barcoding Beads Mix Barcoding Beads evenly, pipette 110 ⁇ L Barcoding Beads (1RXN) into a 1.5mL centrifuge tube, centrifuge briefly, place on a magnetic stand, and let stand for 1min. After the solution is clarified, carefully remove the supernatant liquid. When cleaning, remove the centrifuge tube from the magnetic stand, add 1mL PBS, place it on the magnetic stand after a short centrifugation, and let it stand for 1 min. After the solution is clear, carefully suck off the supernatant and wash 3 times.
- the recommended number of cells is at least 30,000; if the cell diameter is about 15 ⁇ m, the recommended number of cells is at least 20,000; if the cell diameter is about 20 ⁇ m, the recommended number of cells is 10,000-15,000.
- step 2 Repeat the washing process of step 1 for 2-3 times, and use a pipette to pump 100% absolute ethanol back and forth in the chip until no bubbles appear in the chip.
- step 4 Repeat the washing process of step 3 twice, keep a small amount of liquid at the sample outlet, and finally cover the petri dish, and let it stand at room temperature for later use.
- step 4 Repeat step 4 once to wash away the cells that remain on the surface and do not fall into the microwells.
- step 3 Repeat step 3 1-3 times until the excess Barcoding Beads are rinsed off.
- Lysis Buffer is relatively viscous and prone to air bubbles. Be careful not to inject air bubbles into the chip when adding samples.
- the next step of reverse transcription can be performed directly, or the chip can be stored at -80°C for 2 days.
- step 4 Repeat step 4 twice to collect Barcoding Beads that capture mRNA.
- step 6 centrifuge the centrifuge tube briefly, and remove the residual liquid with a 20 ⁇ L pipette, leaving only the Barcoding Beads at the bottom of the centrifuge tube.
- purification magnetic beads Ampure XP purification magnetic beads (hereinafter referred to as purification magnetic beads) were taken out from 4°C 30 minutes in advance and returned to room temperature.
- the purified magnetic beads should be mixed well before use.
- step 3 for a total of 2 rinses.
- the sample can be stored at 4°C for 72 hours, and at -20°C for one week, or it can be directly subjected to QC and quantification after cDNA amplification and purification.
- Qualified cDNA should meet the following conditions at the same time: the size of the main peak fragment should be around 900-2000bp;
- the secondary purification ratio of cDNA should not be lower than 0.6X.
- LA Mix is relatively viscous, and the exact volume should be injected when drawing (it is recommended to add AFI to the system separately when configuring multiple reaction systems). Put the PCR tube of the previous reaction on ice, and configure the following reaction system:
- the purified magnetic beads are relatively viscous, pipette the corresponding volume accurately, otherwise the length of the sorted fragments may be inconsistent with the expectation.
- step 3 for a total of 2 rinses.
- Samples can be stored at -20°C for one week after this step.
- LP Adapter Mix includes 1-8 types, just choose any one.
- the number of amplification cycles should be selected according to the amount of cDNA input, and the selection requirements are as follows:
- the purified magnetic beads are relatively viscous, pipette the corresponding volume accurately, otherwise the length of the sorted fragments may be inconsistent with the expectation.
- step 5 for a total of 2 rinses.
- the range of the main peak fragment must be 300-600bp, 900bp-5000bp should account for less than 20%, and the proportion below 300bp should be less than 20%. If there is a small spike around 200bp, it can be purified at 0.8X and sent for testing.
- Libraries that pass the quality inspection can be directly sent for sequencing, or stored in a -20°C or -80°C refrigerator for 3 months.
- the magnetic beads (purification magnetic beads) were taken out from 4°C 30 minutes in advance and returned to room temperature.
- the magnetic beads are relatively viscous, pipette the corresponding volume accurately, otherwise the length of the sorted fragments may be inconsistent with the expectation.
- samples can be stored at -20°C for 24 hours, or quantified directly after cDNA circularization and purification.
- the magnetic beads (purification magnetic beads) were taken out from 4°C 30 minutes in advance and returned to room temperature.
- the magnetic beads are relatively viscous, pipette the corresponding volume accurately, otherwise the length of the sorted fragments may be inconsistent with the expectation.
- the samples can be stored at 4°C for 72 hours or at -20°C for one week, or the first round of enrichment products can be quantified directly.
- step 3 Pipette 7.5 ⁇ L (0.15x product volume) of magnetic beads into the supernatant collected in step 2), vortex or pipette 10 times to mix well, and incubate at room temperature for 5 minutes.
- step 6) Repeat step 5) for a total of 2 rinses.
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Abstract
本发明提供了用于单细胞分析的方法,包括将细胞和附着有多个条形码寡核苷酸的珠子划分成一个分区。多个条形码寡核苷酸中的每个条形码寡核苷酸可包含细胞条形码和唯一分子标识符(UMI),通过寡核苷酸末尾的polyT序列,与mRNA的polyA序列进行结合,完成mRNA的捕获。通过反转录和PCR扩增,分别完成mRNA的一链和二链合成。将一部分cDNA用于构建转录组测序文库,另一部分cDNA通过环化,形成环状双链cDNA,以此为模板,进行靶基因的特异性富集。由于两部分的cDNA携带的cell barcode信息是匹配的,通过测序之后的Barcode序列匹配,就能获得细胞的表达谱信息和配对的靶基因序列信息。
Description
本申请总体上涉及分子生物学领域,具体提供包括用于高通量单细胞靶标测序的方法、成分、试剂盒和***。
单细胞转录组测序是近年来生物学领域最流行的技术。其超高分辨率可实现对样品信息的准确分析,在生物学的诸多领域具有巨大的应用潜力。例如,研究肿瘤的异质性对疾病发展和药物干预的重要影响,用于解析免疫受体多样性,并转化应用于疫苗接种、癌症和自身免疫疾病。人类和其他脊椎动物的细胞表型多样性可能源于复杂的基因重排和替代RNA剪接事件
[1,2],然而,单细胞转录组测序,受限于目前的短读长mRNA建库测序技术,对于转录本的3’端或者5’端存在明显的选择性,不能有效捕获距离3’端或者5’端较远的基因序列,对于检测复杂的基因重排和RNA剪切就更加困难。
例如,免疫***中每个新分化的T或B淋巴细胞都携带不同的抗原受体,DNA重排改变了T细胞或B细胞抗原受体mRNA 5’端的450个核苷酸
[3]。在B淋巴细胞中,它们使用额外DNA重排在重链mRNA的3’端包含1000-1500个核苷酸的九个替代恒定区序列之间“同种型转换”
[4],并使用替代mRNA剪接来改变IGH mRNA 3’末端的核苷酸以分泌作为抗体的编码受体
[5]。同样,复杂的基因重排和选择性剪接事件在癌细胞中产生病理细胞多样性。因此,迫切需要能够以单细胞分辨率捕获在整个mRNA分子长度上发生的这些序列变化的方法,并将该信息与基因表达特征相结合。
目前,已有的单细胞靶向测序的技术方案,如10X Genomics的单细胞靶向测序的产品,基于探针杂交捕获的方法,在原有的单细胞3’端或者5’端转录组文库的基础上,通过探针杂交捕获感兴趣的基因片段。虽然实现了靶基因的富集,但是未能解决对于末端存在偏好的问题,仍然不能捕获距离3’端或者5’端较远的基因。BD Biosciences的靶向测序产品,基于不同基因设计了富集Panel,采用多重PCR的方式对于感兴趣的基因位点,进行PCR扩增从而获取多个基因的表达信息。但是,这一方案基于3’端的mRNA捕获策略,只能对于靶基因的 表达进行定量,不能获得靶基因的变异信息。对于基因重排后的检测,例如,免疫受体序列,已有一些用于检测单细胞免疫受体的方法和试剂。例如,基于SMART-Seq2捕获免疫受体序列,但是上述的方法依赖于流式细胞仪进行细胞分选检测通量小,通常是10-100个细胞
[6],单个细胞的检测成本均比较高昂,且不能同时检测BCR或转录本。因此,需要开发一款高通量检测单个细胞靶基因(包括但不限于免疫受体核酸序列)的方法来实现对海量单细胞靶向序列的富集。
发明内容
本申请提供了用于高通量单细胞靶标测序的方法、成分、试剂盒和***。
本申请提供了一种在单细胞水平分析细胞基因表达水平以及靶基因序列的方法,其中,所述方法包括:
a.将细胞和附着有多个条形码寡核苷酸的珠子共同落入同一个微孔,其中,多个条形码寡核苷酸的每个条形码寡核苷酸包括细胞条形码和唯一分子标识符(UMI),其中多个条形码寡核苷酸的第一条形码寡核苷酸每个包含能够结合第一信使核糖核酸(mRNA)靶标的polyA尾的polyT序列;
b.在二链合成之后,一部分cDNA会进行环化,形成环状双链cDNA;
c.环状cDNA被用于进行靶基因的富集;以及
d.匹配分析转录组信息与靶基因信息。
优选地,所述多个条码寡核苷酸序列上涵盖两种不同的测序引物结合位点;其中,所述结合位点可用作PCR引物结合序列以扩增cDNA的序列。
优选地,所述多个条码寡核苷酸序列上涵盖两种不同的测序引物结合位点;其中,所述结合位点可用作测序引物结合位点,进行文库测序。
优选地,所述cDNA环化是通过酶促反应进行的。
优选地,所述cDNA环化的酶是DNA连接酶。
优选地,所述cDNA环化的酶是DNA聚合酶。
优选地,所述靶基因的富集是以环状cDNA为模板,用反向PCR的方式进行富集。
优选地,所述信息获取的方式是基因测序。
优选地,所述匹配分析的方法是通过Cell barcode识别后的配对进行。
优选地,所述核酸靶标包括核糖核酸(RNA)、信使RNA(mRNA)和脱氧核糖核酸(DNA),和/或其中所述核酸靶标包括来自细胞、细胞中和/或细胞表面上的细胞的核酸靶标。
优选地,所述被富集的靶基因来源可以是任意脊椎动物。
图1为全长转录本富集原理图;
图2为cDNA环化及全长TCR富集原理图;
图3为全长转录本扩增的产物质检峰图;
图4为全长TCR扩增的产物质检峰图;
图5为TCR TOP10克隆型;
图6为TCR文库测序质控结果。
单细胞转录组测序结合单细胞靶向测序技术,可以同时分析转录组的细胞类型和靶基因序列信息,包括细胞突变信息,是研究肿瘤细胞发育、靶向药物和基因热点突变之间关系的有力工具。单细胞转录组结合靶向突变可以准确识别发生突变的细胞类型,为临床用药提供参考。同时,它可以动态监测用药过程中突变类型和频率的变化。该技术是通过将一部分双链cDNA进行环化,通过反向PCR技术设计链特异性引物富集距离3’端较远的靶基因,并基于Singleron独特的单细胞微流控***,不仅检测普通的单细胞转录组,还可以获得感兴趣的靶基因以及免疫受体序列。本文所公开的方法不仅可以获得高质量的单细胞转录组数据,还可以根据客户的需求,在比转录组低得多的测序深度上获得感兴趣的热点突变信息。该技术具有以下特点:(1)高通量:可同时检测数千个细胞感兴趣区域的突变;(2)深度定制:可根据客户的不同需求设计相应的富集引物;(3)性价比高:实验程序与单细胞转录组工作流程高度兼容。只需要定制引物序列,构建相应的富集文库即可实现目标区域的捕获。
在以下实施例中更详细地公开了上述实施例的一些方面,其不以任何方式旨在限制本公开的范围。
1.单细胞分选、mRNA捕获、反转录及PCR富集
通过将细胞Loading进入Singleron微流控芯片,根据“泊松分布”的原理 完成单个细胞的分离,利用Barcoding Beads完成单个细胞的mRNA的捕获和标记。Barcoding Beads的Oligo序列包含illumina Read1测序引物序列,细胞标签(Cell Barcode),分子标签(UMI)和PolyT核苷酸序列。
通过Barcoding Beads 5’端的PCR handle序列(适配illumina二代测序平台的测序引物)及反转录过程中添加上的TSO序列,扩增完成全长转录组的富集。
2.全长TCR富集及文库构建
将获得的cDNA先环化,再利用Barcoding Beads上的Universial序列和TCR特异性引物(C区序列)进行PCR扩增,完成全长TCR富集。取50ng全长TCR富集产物进行文库构建。
●全长TCR富集
3.单细胞分选和mRNA捕获
3.1准备Lysis Buffer和Barcoding Beads
1.体系配置:室温解冻“Lysis Buffer,Stock”和“100mM DTT”,涡旋离心然后置于冰上。在冰上按下表格配制Lysis Buffer,涡旋混匀并短暂离心,置于冰上备用。
2.清洗Barcoding Beads:将Barcoding Beads混匀后,吸取110μL Barcoding Beads(1RXN)于1.5mL离心管中,短暂离心后置于磁力架上,静置1min,待溶液澄清后,小心吸除上清液。清洗时需将离心管从磁力架上取下,加入1mL PBS,短暂离心后置于磁力架上,静置1min,待溶液澄清后,小心吸除上清液,清洗3次即可。
3.清洗完成后,加入60μL PBS(1RXN)重悬,室温放置待用。
注:若1h内不使用,需放置于4℃暂存。
3.2准备细胞
用PBS将细胞悬液稀释至1.0×10
5-3.0×10
5cells/mL即可用于芯片实验。
若细胞直径10μm左右,建议细胞个数至少达到30000个;若细胞直径15μm左右,建议细胞个数至少达到20000个;若细胞直径20μm左右,建议细胞个数10000-15000个。
3.3芯片操作
3.3.1芯片前处理
1.将微流控芯片置于干净的培养皿上,用200μL的移液器吸取200μL100%无水乙醇从进样口注入芯片,时间控制在10s,及时移除出样口处液体。
2.重复步骤1的冲洗过程2-3次,可使用移液器在芯片中来回抽吸100%无水乙醇直至芯片中不再出现气泡。
3.移除出样口处液体后,吸取200μL 0.02%PBST(PBS中包含0.02%Tween-20,)从进口处注入芯片,时间控制在10s以内,及时移除出样口处液体。
4.重复步骤3的冲洗过程2次,出样口处保留少量液体,最后盖上培养皿盖,室温静置备用。
3.3.2注入细胞
1.移除进出样口多余液体,加200μL PBS润洗芯片(注入时间控制在10s以内),然后移除出样口及进样口多余液体,重复润洗1次。
2.吸取100μL重悬好的细胞,缓慢匀速(建议30s)注入芯片,立即移除出样口多余液体。
3.静置5min使细胞落入微孔内,静置期间可在显微镜下观察细胞落入微孔情况。
4.待细胞落入微孔内,吸取200μL PBS缓慢匀速(建议30s)注入芯片冲洗掉多余细胞,冲洗完毕后立即吸除进出样口液体。
5.重复1次步骤4,冲洗掉留在表面未落入微孔内的细胞。
注:在显微镜下观察表面黏附的多余细胞是否去除干净,若还有残留,可继续加PBS冲洗(冲洗时应缓慢匀速)。
3.3.3注入Barcoding Beads
1.吸取60μL重悬好的Barcoding Beads,缓慢匀速(建议30s)加入进样口,将Barcoding Beads注入芯片。
2.多次吸取100μL PBS,缓慢匀速(约30s)加入进口,使Barcoding Beads缓慢流动,并及时吸取出样口Barcoding Beads,至达到芯片的另一端,在此期间收集进出样口的多余Barcoding Beads。
3.吸取200μL PBS缓慢匀速注入芯片(建议30s),吸去进出样口多余液体。注:吸、推液体时要保证芯片两端口处有足量的PBS以防止气泡进入芯片。
4.重复1-3次步骤3,至冲洗掉多余的Barcoding Beads。
注:在显微镜下观察多余Barcoding Beads是否去除干净,若还有残留,可继续加PBS冲洗。
注:①在显微镜下观察多余Barcoding Beads是否去除干净,若还有残留,可继续加PBS冲洗。②在没有确定达到要求以前,进出样两端的Barcoding Beads都应回收。
5.显微镜下观察Barcoding Beads掉入孔中的情况,若芯片进口端Barcoding Beads空缺较多,可将回收的Barcoding Beads置于磁力架上,吸除上清液提高Barcoding Beads密度后再次注入到芯片的微孔中Barcoding Beads空缺处,静置10s后再冲洗;同理,若芯片出口端Barcoding Beads空缺较多,可将回收的Barcoding Beads注入到出口槽处,用移液器从进口端将Barcoding Beads吸入空缺处,静置10s后再冲洗。
注:推液体时要保证芯片两端口处有适量的PBS以防止气泡进入芯片。
3.3.4注入Lysis Buffer
吸取100μL Lysis Buffer从进样口缓慢注入芯片,时间约20s,立即移除进出样口多余液体。
注:Lysis Buffer较为粘稠且容易产生气泡,加样时需留意勿将气泡注入芯片。
3.4细胞裂解&RNA捕获
室温静置20min用于裂解细胞并释放mRNA,让Barcoding Beads捕获mRNA。
注:完成捕获后可直接进行下一步反转录,也可将芯片放置在-80℃保存2天。
-80℃保存后,若欲进行下一步取出Barcoding Beads的操作,需将芯片取出,室温平衡静置10min,待芯片内试剂恢复液体状态即可。
3.5取出Barcoding Beads
注:①提前配置Wash Buffer 1和Tween 20的混合物,配置方法为在1mL Wash Buffer 1加入Tween 20,使Tween 20的终浓度为0.02%。
②提前用Nuclease-free Water将10×Wash Buffer稀释至1×。
1.将新格元磁力架置于芯片底部,静置1min,保持磁力架在芯片底部,吸取100μL Wash Buffer 1和Tween 20的混合物,缓慢注入芯片,时间约为1min,及时移除出口处液体。
2.保持新格元磁力架置于芯片底部,用200μL Wash Buffer 1和Tween 20的混合物(同步骤1,Tween 20的终浓度为0.02%)加入到出样口凹槽,用于快速润洗出样口凹面,润洗完毕后立即移除液体。
3.重复步骤2,1次,保证出样口无液体剩余。
4.将新格元磁力架转移置于芯片顶部,静置1min,保持磁力架在芯片顶部,于出样口加200μL Wash Buffer 1和Tween 20的混合物(提前吸取1mL Wash Buffer 1,并加入Tween 20,Tween 20的终浓度为0.02%),将200μL枪头***进样口,吸取200μL液体,收集到的含有Barcoding Beads的液体转移至1.5mL离心管内。
5.重复2次步骤4,收集捕获到mRNA的Barcoding Beads。
4.反转录及扩增
4.1反转录
1.体系配置:提前室温解冻“2×RT Master Mix”,“100mM DTT”和“TS Primer”,涡旋离心然后置于冰上,在冰上按照如下表格配制RT Mix,涡旋混匀并短暂离心(若2×RT Master Mix试剂溶液中有沉淀物,请用手指轻弹试剂管壁,并用移液器轻轻吹打混匀试剂溶液,确保试剂溶液澄清后再使用)。
2.将步骤3.5.5装有Barcoding Beads的离心管短暂离心后置于1.5mL规格磁力架(DynaMag
TM-2磁力架/12321D/Thermo,本小节磁力架同规格)上,待溶液澄清后,小心吸除上清液。
3.从磁力架上取下离心管,用1mL移液器加入1mL Wash Buffer 1,然后短暂离心,置于磁力架上,待溶液澄清后小心吸除上清液。
4.从磁力架上取下离心管,加入500μL 1×Wash Buffer(提前用Nuclease-free Water稀释10×Wash Buffer至1×)混匀后短暂离心,置于磁力架上,待溶液澄清后小心吸除上清液。
5.取下离心管,短暂离心后再置于磁力架上,用20μL的移液器吸取残余的液体。只留下离心管底部的Barcoding Beads。
6.向4.1.5收集产物的离心管中加入200μL配好的RT Mix,并吹吸3下混匀。
7.置于提前设置好的金属浴中,42℃,转速1000rpm,反应90min(提前预热)。
注:42℃反应90min后,若无法立即进行下一步,将反转录产物70℃灭活15min后,可室温放置15h(可在金属浴上过夜)。
4.2 PCR扩增
1.体系配置:提前室温解冻“Amplification Master Mix”、“G Primer Mix”,涡旋离心然后置于冰上,按照如下表格在冰上配制PCR Mix,涡旋混匀并短暂离心。
2.将4.1的反转录产物短暂离心,置于1.5mL规格磁力架(DynaMag
TM-2磁力架/12321D/Thermo,本小节磁力架同规格)上,待溶液澄清后小心吸除上清液。
3.将离心管从磁力架上取下,用1mL移液器加入1mL Wash Buffer 2。
4.混匀后短暂离心,置于磁力架上,待溶液澄清后小心吸除上清液。
5.将离心管从磁力架上取下,用1mL移液器加入1mL Wash Buffer 3。
6.混匀后短暂离心,置于磁力架上,待溶液澄清后小心吸除上清液。
7.将离心管从磁力架上取下,加入1mL Nuclease-free Water,颠倒混匀后短暂离心,置于磁力架上,待溶液澄清后小心吸除上清液。
8.重复1次步骤6,将离心管短暂离心,用20μL移液器移除残余液体,只留下离心管底部的Barcoding Beads。
9.向管中加入400μL PCR Mix,一边吹打混匀,一边分装到8联排管中(防止Barcoding Beads分装不均匀),每管分装液体体积为50μL。
10.盖好8联排管管盖,置于PCR仪中进行扩增,PCR程序见下表。PCR程序结束后,可将样品在4℃保存48h和-20℃保存一周,或者直接进行cDNA扩增纯化。
4.3产物纯化
1.Ampure XP纯化磁珠(后简称纯化磁珠)提前30min从4℃中取出,恢复室温。
注:纯化磁珠使用前需混匀。
将PCR扩增产物收集到1.5mL离心管中,短暂离心,量取体积。加入0.6×纯化磁珠(例如,量取的产物体积为400μL,则加入0.6×400=240μL体积的纯化磁珠)涡旋混匀后,室温孵育5min,短暂离心,置于1.5mL规格磁力架(DynaMag
TM-2磁力架/12321D/Thermo,本小节磁力架同规格)上静置5min;至液体透明澄清,小心吸除上清液至新的1.5mL离心管中,暂留。注:纯化磁珠比较粘稠,准确移取相应的体积,否则可能导致分选的片段长度与预期不一致。
2.保持离心管始终处于磁力架上,加入800μL新配制的80%乙醇漂洗纯化磁珠。室温孵育30s,小心吸除上清液。
3.重复步骤3,共计漂洗2次。
4.取下离心管,短暂离心,再次置于磁力架上,吸去多余酒精,开盖晾干约2min(不要超过5min)。
5.取下离心管,加入20μL Nuclease-free Water,吹打混匀纯化磁珠,室温孵育5min,短暂离心后静置于磁力架上,至液体透明澄清。
6.吸取上清并转移至新的EP管中,即为纯化产物。
注:将样品置于4℃可保存72h,置于-20℃可保存一周,或者可直接进行cDNA扩增纯化后的QC和定量。
4.4扩增纯化产物质检
1.取适量(建议1μL)样品进行cDNA浓度和片段大小检测。
2.质检结果请参照图8-4,合格cDNA应同时满足以下几个条件:主峰片段大小应在900-2000bp左右;1000bp-5000bp占比大于15%;300bp以下片段占比小于40%。
3.cDNA二次纯化(选做)
欲进一步降低cDNA 300bp以下片段所占比例,可按下表根据小片段占比 进行二次纯化:
注:cDNA二次纯化比例不能低于0.6X。
5.文库构建
5.1片段化
1.提前按照下表设置PCR程序,并提前设置PCR仪热盖温度为75℃。
2.室温解冻FR Buffer V2,确保完全融化,涡旋混匀并离心后冰上备用;使用前将FR Mix V2涡旋混匀,置于冰上备用。将灭菌的PCR管置于冰上,配置如下反应体系:
注:a)cDNA总量不足10ng时,将全部cDNA投入进行建库。
b)cDNA总量在10-50ng时,使用10ng投入量进行建库。
c)cDNA总量在50ng以上时,使用50ng投入量进行建库。
3.用移液器轻柔吹打充分混匀,短暂离心后将PCR管置于PCR仪中, 跳过PCR程序第一步。
4.完成反应后立即进行下一步接头连接。
5.2连接接头
1.照下表设置PCR程序,并关闭PCR仪热盖加热后置于冰上备用。
2.LA Mix较粘稠,吸取时注取准确体积(配置多个反应体系时建议将AFI单独加入体系)。将上一步反应的PCR管置于冰上,配置如下反应体系:
3.使用移液器轻柔吹打混匀并短暂离心。将反应管置于PCR仪中,运行程序。
5.3接头连接后产物纯化
1.纯化磁珠提前30min从4℃中取出,恢复室温备用。将产物PCR管短暂离心,使用移液器量产物体积,涡旋混匀纯化磁珠并吸取34.2μL(0.5×产物体积)加入至68.5μL片段化产物中,涡旋振荡充分混匀,室温孵育5min。
注:纯化磁珠比较粘稠,准确移取相应的体积,否则可能导致分选的片段长度与预期不一致。
2.将PCR管短暂离心后置于0.2mL规格磁力架(DynaMag
TM-PCR磁力架/492025/Thermo,本小节磁力架同规格)上,使纯化磁珠与液体分离,待溶液澄清后(约5min),小心移除上清至一个新的无菌PCR管中,暂时留存。
3.保持纯化磁珠所在PCR管置于磁力架上,用200μL新鲜配制的80% 乙醇漂洗纯化磁珠,室温孵育30s,小心吸除上清液。
4.重复步骤3,总计漂洗2次。
5.从磁力架上取下纯化磁珠所在PCR管,短暂离心,再次置于磁力架上,吸去管内及管壁多余酒精,开盖晾干纯化磁珠1min(不要超过2min)。
6.将纯化磁珠所在PCR管从磁力架上取下,加入17μL 0.1×TE(使用Nuclease-free Water按1:10稀释1×TE),涡旋振荡或吹打混匀纯化磁珠,室温孵育5min。
7.将纯化磁珠所在PCR管短暂离心后置于磁力架上,使纯化磁珠与液体分离,待溶液澄清后(约5min),小心吸取15μL上清至新的灭菌PCR管中用于步骤5.4 PCR富集。
注:此步骤结束后可以将样品于-20℃保存一周。
5.4 PCR富集
1.将PCR管置于冰上,配置如下反应体系,并使用移液器轻柔吹打混匀并短暂离心,将反应管置于PCR仪中。
LP Adapter Mix包括1-8种,任意选择一种即可。
2.提前按照下表设置PCR程序,并使PCR仪热盖保持在105℃
扩增循环数需按cDNA投入量进行选择,选择要求如下:
5.5扩增产物片段分选
1.纯化磁珠提前30min从4℃中取出,恢复室温备用。将5.3.7产物PCR管短暂离心,使用移液器量产物体积,涡旋混匀纯化磁珠并吸取25μL(0.5×产物体积)加入至50μL产物中(如体积不足50μL使用Nuclease-free Water补足),涡旋振荡充分混匀,室温孵育5min。
注:纯化磁珠比较粘稠,准确移取相应的体积,否则可能导致分选的片段长度与预期不一致。
2.将PCR管短暂离心后置于0.2mL规格磁力架(DynaMag
TM-PCR磁力架/492025/Thermo,本小节磁力架同规格)上静置,使纯化磁珠与液体分离,待溶液澄清后(约5min),小心转移上清至一个新的无菌PCR管中,丢弃纯化磁珠。
3.涡旋振荡混匀纯化磁珠并吸取7.5μL(0.15×产物体积)加入至上清中,涡旋振荡充分混匀,室温孵育5min。
4.将PCR管短暂离心后置于磁力架上,使纯化磁珠与液体分离,待溶液澄清后(约5min),小心转移上清液置于新的PCR管暂时留存。
5.保持PCR管始终处于磁力架上,加入200μL新鲜配制的80%乙醇漂洗纯化磁珠。室温孵育30s,小心吸除上清液。
6.重复步骤5,总计漂洗2次。
7.从磁力架上取下PCR管,短暂离心,再次置于磁力架上,吸去多余酒精,开盖晾干纯化磁珠1min(不要超过2min)。
8.将PCR管从磁力架上取出,加入20μL Nuclease-free Water洗脱。涡旋振荡或吹打混匀纯化磁珠,室温孵育5min。
9.将PCR管短暂离心并置于磁力架上,使纯化磁珠与液体分离,待溶液澄清后(约5min)小心吸取上清至新的灭菌1.5mL离心管中,-20℃或-80℃保存三个月。
5.6文库质检
1.取适量(建议1μL)样品进行产物浓度和片段大小检测。
2.主峰片段范围须在300-600bp,900bp-5000bp占比小于20%,300bp以下占比小于20%。若存在200bp左右小尖峰凸起,可0.8X纯化后送测。
3.质检合格的文库,可直接送测序,也可置于-20℃或-80℃冰箱保存3个月。
6.全长免疫受体(TCR)富集
6.1全长免疫受体(TCR)cDNA环化
1.取250ng cDNA产物,将PCR管置于冰上按照如下表格配制环化mix,涡旋混匀并短暂离心。
2.使用移液器轻柔充分混匀,短暂离心后将反应管置于PCR仪中,PCR仪根据下表设置并运行反应,PCR仪热盖85℃:
注:环化完成后建议立即纯化。
3.产物纯化
1)磁珠(纯化磁珠)提前30min从4℃中取出,恢复室温。
注:磁珠使用前需混匀。
2)将PCR管中的液体,瞬离,计算体积。加入1.3×产物体积的磁珠,吹打混匀后,室温孵育5min,短暂离心,置于磁力架上静置5min;至液体透明澄清,小心移除上清至新的PCR管中,暂留。
注:磁珠比较粘稠,准确移取相应的体积,否则可能导致分选的片段长度与预期不一致。
3)保持PCR管始终处于磁力架上,加入200μL新配制的80%乙醇漂洗磁珠。室温孵育30s,小心移除上清。
4)重复3),共计漂洗两次。
5)取下PCR管,短暂离心,再次置于磁力架上,吸去多余酒精,晾干。
6)取下PCR管,加入20μL Nuclease-free Water,吹吸混匀磁珠,室温孵育5min,短暂离心后静置于磁力架上,至液体透明澄清。
7)吸取上清并转移至新的EP管中,即为纯化产物。
安全暂停点:将样品置于-20℃可保存24h,或者直接进行cDNA环化纯化后的定量。
4.环化纯化产物质检
取1μL样品进行Qubit浓度检测。
6.2全长免疫受体(TCR)第一轮富集
1.取20ng环化产物,将PCR管置于冰上按照如下表格配制第一轮富集PCR mix,涡旋混匀并短暂离心。
2.使用移液器轻柔充分混匀,短暂离心后将反应管置于PCR仪中,PCR仪根据下表设置并运行反应,PCR仪热盖105℃:
3.产物纯化
1)磁珠(纯化磁珠)提前30min从4℃中取出,恢复室温。
注:磁珠使用前需混匀。
2)将PCR管中的液体,瞬离,计算体积。加入0.6×产物体积的磁珠,吹打混匀后,室温孵育5min,短暂离心,置于磁力架上静置5min;至液体透明澄清,小心移除上清至新的PCR管中,暂留。
注:磁珠比较粘稠,准确移取相应的体积,否则可能导致分选的片段长度与预期不一致。
3)保持PCR管始终处于磁力架上,加入200μL新配制的80%乙醇漂洗磁珠。室温孵育30s,小心移除上清。
4)重复3),共计漂洗两次。
5)取下PCR管,短暂离心,再次置于磁力架上,吸去多余酒精,晾干。
6)取下PCR管,加入20μL Nuclease-free Water,吹吸混匀磁珠,室温孵育5min,短暂离心后静置于磁力架上,至液体透明澄清。
7)吸取上清并转移至新的EP管中,即为纯化产物。
注:将样品置于4℃可保存72h或在-20℃保存一周,或者直接进行第一轮富集产物的定量。
4.扩增纯化产物质检
取1μL样品进行Qubit浓度检测。
6.3全长免疫受体(TCR)第二轮富集
1.取20ng第一轮富集产物,将PCR管置于冰上按照如下表格配制第一轮富集PCR mix,涡旋混匀并短暂离心。
2.使用移液器轻柔充分混匀,短暂离心后将反应管置于PCR仪中,PCR仪根据下表设置并运行反应,PCR仪热盖105℃:
3.产物纯化
1)提前30min取出纯化磁珠,恢复室温,备用。将PCR管瞬离,量取PCR产物体积,加入25μL磁珠(0.5×产物体积),涡旋振荡或使用移液器吹打10次充分混匀,室温孵育5min。
2)将PCR管短暂离心后置于磁力架上,使磁珠与液体分离,待溶液澄清后(约5min),小心转移上清至一个新有无菌PCR管中。
3)吸取7.5μL(0.15x产物体积)磁珠至步骤2)收集的上清中,涡旋振荡或使用移液器吹打10次充分混匀,室温孵育5min。
4)将PCR管短暂离心后置于磁力架上,使磁珠与液体分离,待溶液澄清后(约5min),小心移除上清。
5)保持PCR管始终处于磁力架上,加入200μL新鲜配制的80%乙醇漂洗磁珠。室温孵育30s,小心移除上清。
6)重复步骤5),总计漂洗2次。
7)从磁力架上取下PCR管,短暂离心,再次置于磁力架上,吸去多余酒精,开盖使磁珠暴露于空气中干燥2min。
8)将PCR管从磁力架上取出,加入20μL Nuclease-free Water洗脱。涡旋振荡使磁珠和液体充分混匀,室温孵育5min。
9)将PCR管短暂离心并置于磁力架上,使磁珠与液体分离,待溶液澄清后(约5min)小心吸取18μL上清至新的灭菌PCR管中。
4.扩增纯化产物质检
1)取1μL样品进行Qubit浓度检测。
2)取2μL样品进行片段大小的检测。
注意:样品浓度过高时需要将样品稀释到3-5ng/μL,防止浓度过高,影响质检果。人源质检结果请参照图10-1,显示一条特异条带,在700bp左右。鼠 源质检结果请参照图10-2,显示一条特异条带,800bp左右。
参考文献:
[1]V(D)J Recombination and the Evolution of the Adaptive Immune System[J].PLoS Biology,2003,1(1):e16-.
[2]Eric T,Wang,Rickard,et al.Alternative isoform regulation in human tissue transcriptomes.[J].Nature,2008.
[3]Bassing C H,Swat W,Alt F W.The Mechanism and Regulation of Chromosomal V(D)J Recombination[J].Cell,2002,109(2):S45-S55.
[4]Chaudhuri J,Alt F W.Class-switch recombination:interplay of transcription,DNA deamination and DNA repair[J].Nature Reviews Immunology.
[5]Frederick,W,Alt,et al.Synthesis of secreted and membrane-bound immunoglobulin mu heavy chains is directed by mRNAs that differ at their 3′ends[J].Cell,1980,20(2):293-301.
[6]Picelli,S.et al.Smart-seq2for sensitive full-length transcriptome profiling in single cells.Nat.Methods 10,1096–1098,(2013).
Claims (11)
- 一种在单细胞水平分析细胞基因表达水平以及靶基因序列的方法,其包括:a.将细胞和附着有多个条形码寡核苷酸的珠子共同落入同一个微孔,其中,多个条形码寡核苷酸的每个条形码寡核苷酸包括细胞条形码和唯一分子标识符(UMI),其中多个条形码寡核苷酸的第一条形码寡核苷酸每个包含能够结合第一信使核糖核酸mRNA靶标的polyA尾的polyT序列;b.在二链合成之后,一部分cDNA会进行环化,形成环状双链cDNA;c.环状cDNA被用于进行靶基因的富集;以及d.匹配分析转录组信息与靶基因信息。
- 根据权利要求1所述的方法,其中,所述多个条码寡核苷酸序列上涵盖两种不同的测序引物结合位点;其中,所述结合位点可用作PCR引物结合序列以扩增cDNA的序列。
- 根据权利要求1所述的方法,其中,所述多个条码寡核苷酸序列上涵盖两种不同的测序引物结合位点;其中,所述结合位点可用作测序引物结合位点,进行文库测序。
- 根据权利要求1所述的方法,其中,所述cDNA环化是通过酶促反应进行。
- 根据权利要求4所述的方法,其中,所述cDNA环化的酶是DNA连接酶。
- 根据权利要求4所述的方法,其中,所述cDNA环化的酶是DNA聚合酶。
- 根据权利要求1所述的方法,所述靶基因的富集是以环状cDNA为模板,用反向PCR的方式进行富集。
- 根据权利要求1所述的方法,其中,所述信息获取的方式是基因测序。
- 根据权利要求1所述的方法,其中,所述匹配分析的方法是通过Cell barcode识别后的配对进行。
- 根据权利要求1所述的方法,其中,所述核酸靶标包括核糖核酸RNA、信使核糖核酸mRNA和脱氧核糖核酸DNA,和/或其中所述核酸靶标包括来自细胞、细胞中和/或细胞表面上的细胞的核酸靶标。
- 根据权利要求1所述的方法,所述被富集的靶基因来源可以是任意脊椎动物。
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