WO2017084023A1 - 一种高通量的单细胞转录组建库方法 - Google Patents

一种高通量的单细胞转录组建库方法 Download PDF

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WO2017084023A1
WO2017084023A1 PCT/CN2015/094777 CN2015094777W WO2017084023A1 WO 2017084023 A1 WO2017084023 A1 WO 2017084023A1 CN 2015094777 W CN2015094777 W CN 2015094777W WO 2017084023 A1 WO2017084023 A1 WO 2017084023A1
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reagent
cell
nanoliters
amplification
reaction
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French (fr)
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王磊
李贵波
李阳
林欣欣
王晶晶
吴靓
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深圳华大基因研究院
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Priority to PCT/CN2015/094777 priority Critical patent/WO2017084023A1/zh
Priority to CN201580082867.9A priority patent/CN108138365B/zh
Publication of WO2017084023A1 publication Critical patent/WO2017084023A1/zh

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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms

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  • the invention relates to the technical field of transcriptome library construction of cells, in particular to a high-throughput single-cell transcriptome database building method.
  • the invention provides a high-throughput single-cell transcriptome database construction method, which has the characteristics of high-flux, low-cost and integration, and can realize automation and scale of single-cell construction.
  • the high-throughput single-cell transcriptome construction method of the present invention comprises: dispensing a cell suspension having a predetermined cell density into each microwell of a nano-upgraded microwell chip using a nano-scaled micro-dispensing platform, using cells The lysate lyses the cells in the micropores; the reverse transcription reagent is distributed into the micropores for reverse transcription reaction using the above micro-dispensing platform; and the cDNA amplification reagent is distributed into the micropores by using the above micro-dispensing platform; The cDNA amplification reaction was carried out, and the single cell amplification was monitored by a fluorescence quantitative PCR instrument.
  • 1 is an amplification dissolution curve of a partial sample according to an embodiment of the present invention, wherein the upper graph shows the relationship between the fluorescence intensity and the number of cycles, the lower graph shows the relationship between the fluorescence intensity and the temperature, and the curve a shows the positive control, the curve b A negative control is shown, curves c, d are shown as two single cell samples;
  • Figure 3 is a diagram showing the number of genes detected in a single cell sample according to an embodiment of the present invention, wherein libraries 1, 2, 3, and 4 represent a single cell library, and rpkm represents a map to a display per 1 million maps of reads. Reads Per Kilobase of exon model per Million mapped reads.
  • the high-throughput single-cell transcriptome construction method of the present invention is implemented based on a micro-dispensing platform, because the miniaturization of the sample is critical to achieve single-cell distribution.
  • the liquid separation capacity of the micro-dispensing platform has achieved a breakthrough in nano-upgrading, which is a qualitative leap compared with the traditional micro-upgraded micro-dispensing platform.
  • the invention realizes the single cell distribution based on the nano-dispensing micro-dispensing platform, and realizes a series of in-situ cell lysis, reverse transcription reaction and cDNA amplification reaction, and completes the single cell transcriptome establishment.
  • the significant advantages of the present invention are reflected in high throughput, low cost, and integration over the prior art.
  • the so-called high throughput means that the present invention is capable of obtaining a large number of single cells at a time compared to the prior art, especially based on microfluidic chip technology, and it has been confirmed that a single chip can complete a library construction of more than 1000 single cells at a time.
  • low cost is meant that the average cost of library construction per single cell is significantly reduced due to the simplicity and high throughput of the method of the invention.
  • the so-called integration means that all steps after the completion of single cell distribution are integrated on one chip.
  • a nanoscale micro-dispensing platform that can be used in the present invention such as the SmartChip (TM) MultiSample NanoDispenser (MSND) from Wafergen Biosystems, can be referred to as a SmartChip (TM) multi-sample nano-dispensing dispenser.
  • TM SmartChip
  • MSND MultiSample NanoDispenser
  • the microporous chip used in the present invention is a nanoscale microporous chip, which is essential for the separation of single cells and the subsequent reactions.
  • Nanoscale microchips that can be used such as the SmartChip from Wafergen Biosystems, can be tailored to various nanoscale capacities, and each microwell of a typical microporous chip useful in the present invention has a volume of 350 nL.
  • a real-time PCR instrument is used to assist in monitoring the amplification of single cells, and to determine single cell microwells based on the amplification.
  • Useful in the present invention are quantitative PCR instrument, e.g. Wafergen Biosystems company SmartChip TM Real-Time PCR Cycler, may be referred to SmartChip TM real-time PCR cycler.
  • the method of the invention can realize single cell separation, cell lysis, reverse transcription reaction (RT) and cDNA amplification reaction in one step in the micropore chip, and optionally, TN5 transposase reagent can be used for fragmentation treatment, if necessary, And steps such as PCR library amplification.
  • a single microwell chip can complete a library construction of more than 1000 single cells at a time.
  • the amount of reagent per reaction can be limited to nanoscale upgrades. For example, in one embodiment of the invention, the amount of reagent per reaction is about 300 nL, which greatly reduces the cost of the experiment.
  • transcriptome sequencing showed that the library constructed by this method has high detection sensitivity and good reproducibility, and at least 10,000 genes can be detected per single cell library.
  • the successful development of the method of the invention can effectively improve the speed of RNA-seq construction at a single cell level, reduce manual operation, save high reagent costs, and realize automation and scale of single cell construction.
  • controlling the density of the cell suspension is critical for efficiently dispensing single cells into the microwells of the microwell chip.
  • the inventors have found that in the case where the cells are uniformly distributed, when the cell density is 4/ ⁇ L or less, the number of cells in 98% or more of the micropores is not more than one.
  • the cell density is 2-4 cells/ ⁇ l, and if the cell density is less than 2 cells/ ⁇ l, there may be too many cells in the micropore that are not distributed, and the single cell is reduced. Effective distribution rate; when the cell density is greater than 4 cells/ ⁇ l, there may be too many cells distributed to more than one cell, which will correspondingly reduce the effective distribution rate of single cells.
  • the cell suspension, the cell lysate, the reverse transcription reagent and the cDNA amplification reagent are all distributed into the micropores through the nano-dispensing micro-dispensing platform, and after a large number of experimental studies, it is found that the tens of nanometer upgrade Other dosages can achieve better results, such as cell suspensions, cell lysates, reverse transcription reagents, and cDNA amplification reagents that are dispensed into the microwells can range from 35 nanoliters to 50 nanoliters.
  • the MSND of Wafergen Biosystems is used as a micro-dispensing platform having a minimum liquid separation volume of 35 nanoliters, so in this preferred embodiment, the cells are dispensed into the microwells.
  • the volume of the liquid, cell lysate, reverse transcription reagent and cDNA amplification reagent was 35 nanoliters.
  • the volume of the micropores is not less than 350 nanoliters.
  • the amount of the above reagents can fluctuate within a range of several tens of nanometers, but too much or too little reagent usage may result in a decrease in the effectiveness of the present invention. If the dosage exceeds 100 nanoliters, the single cell separation effect may not be good, and the amount of the reaction reagents in the subsequent steps will also increase, thereby increasing the cost. Therefore, in the present invention, for a cell suspension, a cell lysate, a reverse transcription reagent, and a cDNA amplification reagent, a reagent amount of 100 nanoliters or more or more is not recommended.
  • the present invention is a liquid separation operation for a very small amount of reagents, which may contain air bubbles, and bubbles may be difficult to remove in a very small amount of reagents, it is preferable to distribute the cell suspension, reverse transcription reagent or cDNA amplification reagent. After reaching the micropores, the bubbles were removed by centrifugation. If the cDNA amplification reaction is completed, other reactions are carried out, and it is also preferable to remove the bubbles by centrifugation.
  • the invention uses a real-time PCR instrument to assist in monitoring single cell expansion. If total RNA is used as a positive control and cell supernatant is used as a negative control, the typical single-cell amplification product is marked by a single peak with a Tm peak value greater than or equal to 88 ° C, and a Ct value between the positive control and the negative. Between the values of the comparison. To further improve the accuracy of single cell selection, it is possible to remove high or low Ct values, such as samples with minimum and maximum Ct values of 10%.
  • the transcriptome library of single cells has been constructed after cell lysis, reverse transcription reaction and cDNA amplification reaction. However, in practical applications, subsequent reactions are usually performed.
  • the TN5 transposase reagent is dispensed into the microwells using a micro-dispensing platform to fragment the product of the cDNA amplification reaction and then terminate The reaction reagent is dispensed into the micropore to terminate the reaction; thereafter, the PCR amplification reaction reagent is dispensed into the microwell using a micro-dispensing platform to perform PCR library amplification on the fragmented product.
  • the amount of the TN5 transposase reagent may be from 100 nanoliters to 150 nanoliters; the amount of the terminating reagent may be 35 nanoliters. Up to 50 nanoliters; PCR amplification reagents can be used in amounts ranging from 50 nanoliters to 75 nanoliters.
  • the TN5 transposase reagent is used in an amount of 100 nanoliters; the amount of the terminating reagent is 35 nanoliters; and the amount of the PCR amplification reagent is 50 nanoliters.
  • the primer pair comprises a 5' end primer and a 3' end primer, the difference between the different 5' end primers or the difference between the different 3' end primers is only in the respective
  • the tag sequence may be a random sequence of a particular length N (such as N being a natural number of 6-10, preferably 8).
  • the tag sequence can be located at various positions of the primer. It is preferably located at the middle of the primer.
  • the method of the invention may further comprise, after amplification of the PCR library: aspirating the products in the single cell wells assisted by the PCR instrument and mixing them together. Further, the mixed products can be purified and selected using a nucleic acid purification kit to select a nucleic acid fragment of a predetermined length.
  • This embodiment includes a series of steps such as single cell preparation, single cell lysis, reverse transcription (RT) reaction, cDNA amplification reaction, and amplification of TN5 transposase.
  • the reagent loading and PCR operations were performed in accordance with Wafergen Biosystems' related instrument instructions.
  • the TN5 transposase was prepared according to the method published by Simone Picelli et al. (Genome Res. 2014. 24: 2033-2040).
  • Single cell library preparation was performed using the Hela S3 cell line, and adherent Hela cells were digested with trypsin, and 1 mL of the digested cell suspension was taken. The cells were centrifuged at 1000 rpm for 5 min, resuspended in 1 x PBS, and repeated once. The Percoll stock solution was mixed with 10 x PBS at a volume ratio of 9:1 to form an isotonic solution of the cells. The resuspended cells were added to a configured 20% isotonic solution, and the cell density was adjusted to 4 / ⁇ L to complete cell sample preparation.
  • the volume of the chip is 35nL per well. Seal with a membrane, centrifuge at 2600 rcf, 12 ° C for 5 min to remove air bubbles.
  • Reverse transcription reagent component Chip hole usage 200U/ ⁇ L SSII 4.2 5 ⁇ SuperScript II First-Strand Buffer 11.2 5M Betaine (betaine) 11.2 100mM MgCl 2 5.04 100mM DTT 1.4 100 ⁇ M TSO 0.56 40U/ ⁇ L RNAse inhibitor 1.4 Total amount 35
  • TSO sequence 5'-AAGCAGTGGTATCAACGCAGAGTACATrGrG+G-3' (SEQ ID NO: 2, rG represents riboguanosines, +G represents locked nucleic acid modification).
  • Primer A 5'-CTGTCTCTTATACACATCT-3' (SEQ ID NO: 4);
  • Primer B 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3' (SEQ ID NO: 5);
  • Primer C 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3' (SEQ ID NO: 6).
  • reaction 1 and reaction 2 Vortex and mix thoroughly, and centrifuge briefly to return the solution to the bottom of the tube. Placed in the PCR machine, the following reaction procedure was carried out: 75 ° C for 15 min; 60 ° C for 10 min; 50 ° C for 10 min; 40 ° C for 10 min; 25 ° C for 30 min.
  • reaction 1 and the reaction 2 are mixed in an equal volume and mixed. Name it Adapter Mix and store at -20 °C.
  • reaction was placed at 25 ° C for 60 min.
  • the reaction product was named as a fragmented enzyme advanced mixture V5S (Tagment Enzyme Advanced Mix V5S) and stored at -20 °C.
  • Primer 1 sequence 5'-AATGATACGGCGACCACCGA-3' (SEQ ID NO: 8);
  • Primer 2 sequence 5'-CAAGCAGAAGACGGCATACGA-3' (SEQ ID NO: 9).
  • Amplification reagents were prepared (Table 8). A N7XX primer was added to each well of a 384-well plate for uniform mixing. MSND platform selection (72 ⁇ 72 mode) was applied. 50 ⁇ L of each well was loaded in the chip, 2600 rcf, and centrifuged at 12 ° C for 5 min. , remove bubbles.
  • the chip was placed in a thermal cycler for amplification.
  • the wells that have been successfully amplified by single cells are determined, and pooled together using a glass needle having a diameter of less than 200 ⁇ m or a special pipette.
  • the pooled product was selectively purified using Agencourt AMPure XP beads.

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Abstract

本发明提供了一种高通量的单细胞转录组建库方法，包括：采用纳升级的微量分液平台，将具有预定细胞密度的细胞悬液分配到纳升级的微孔芯片的每个微孔中；用细胞裂解液裂解所述细胞；将反转录试剂分配到所述微孔中进行反转录反应；将cDNA扩增试剂分配到所述微孔中进行cDNA扩增反应，同时采用荧光定量PCR仪监测单细胞扩增情况。

Description

一种高通量的单细胞转录组建库方法 技术领域
本发明涉及细胞的转录组文库构建技术领域，尤其涉及一种高通量的单细胞转录组建库方法。
背景技术
随着基因测序技术水平的提高以及重大国际合作项目的相继开展，基因组研究发展越来越快。2011年，《自然方法》杂志(Nature Methods)将单细胞测序列为年度值得期待的技术之一。2013年，《科学》杂志(Science)将单细胞测序列为年度最值得关注的六大领域榜首。这些都表明，单细胞测序已逐渐成为科研热点，有望成为近年最值得关注的测序技术。
在单细胞研究工作中，扩大试验规模是确保采集足够多的生物多样性信息的关键。使用微流控芯片虽然可以将单个细胞分离，并进行样品扩增，但是通量不高。其它针对单细胞的方法均存在很多缺陷，导致检测灵敏度不高、基因表达信息丢失严重、技术噪音高、操作失误率高、重复性差，而且针对数以千计甚至万计的细胞数量时，实验费用又成为了最大的瓶颈，高昂的单细胞扩增的费用导致单细胞扩增仍然处于不成熟的阶段，在技术上和成本上还远没有达到大规模应用的地步。
发明内容
本发明提供一种高通量的单细胞转录组建库方法，具有高通量、低成本、一体化的特点，能够实现单细胞建库的自动化和规模化。
本发明的高通量的单细胞转录组建库方法，包括：采用纳升级的微量分液平台将具有预定细胞密度的细胞悬液分配到纳升级的微孔芯片的每个微孔中，用细胞裂解液裂解上述微孔内的细胞；采用上述微量分液平台将反转录试剂分配到上述微孔中进行反转录反应；采用上述微量分液平台将cDNA扩增试剂分配到上述微孔中进行cDNA扩增反应，同时采用荧光定量PCR仪辅助监测单细胞扩增情况。
附图说明
图1为本发明实施例中部分样品的扩增溶解曲线，其中上图示出了荧光强度与循环数的关系，下图示出了荧光强度与温度的关系，曲线a显示阳性对照，曲线b显示阴性对照，曲线c、d显示为两个单细胞样品；
图2为本发明实施例中部分样品的Agilent 2100检测结果；
图3为本发明实施例中单细胞样品中检测的基因数目，其中文库1、2、3、4分别代表一个单细胞的文库，rpkm表示每1百万个map上的reads中map到外显子的每1K个碱基上的reads个数(Reads Per Kilobase of exon model per Million mapped reads)。
具体实施方式
下面通过具体实施方式结合附图对本发明作进一步详细说明。
本发明的高通量的单细胞转录组建库方法是基于微量分液平台实现的，因为要实现单细胞的分配，样品的微量化非常关键。目前，微量分液平台的分液能力已经实现了纳升级的突破，相比传统的微升级的微量分液平台，是质的飞跃。本发明正是以纳升级的微量分液平台为基础实现单细胞的分配，进而实现一系列的原位细胞裂解、反转录反应和cDNA扩增反应等，而完成单细胞转录组建库。
相比现有技术而言，本发明的显著优势体现在高通量、低成本和一体化。所谓高通量是指，相比现有技术尤其是基于微流控芯片的技术，本发明能够一次性获得大量的单细胞，已经证实单个芯片一次即可完成大于1000个单细胞的文库构建。所谓低成本是指，由于本发明方法的简单易行及高通量的特点，而显著降低了每个单细胞的文库构建的平均成本。所谓一体化是指，单细胞分配完成以后的所有步骤都集成于一块芯片上进行。
可用于本发明中的纳升级的微量分液平台，例如Wafergen Biosystems公司的SmartChipTMMultiSample NanoDispenser(MSND)，可以称作SmartChipTM多样品纳升级分配器。
本发明中配合使用的微孔芯片是纳升级的微孔芯片，这对于单细胞的分离以及其后的各种反应的进行至关重要。可以采用的纳升级的微孔芯片，例如Wafergen Biosystems公司的SmartChip，可以定制成各种纳升级的容量，可用于本发明的典型的微孔芯片的每个微孔具有350nL的容积。
荧光定量PCR仪用于辅助监测单细胞的扩增情况，根据扩增情况来判断单 细胞微孔。可用于本发明的荧光定量PCR仪，例如Wafergen Biosystems公司的SmartChipTMReal-Time PCR Cycler，可以称作SmartChipTM实时PCR循环仪。
本发明的方法能够在微孔芯片中一步实现单细胞分离、细胞裂解、反转录反应(RT)以及cDNA扩增反应，根据需要还可以任选地使用TN5转座酶试剂进行片段化处理，以及PCR文库扩增等步骤。使用本发明的方法，单个微孔芯片一次即可完成大于1000个单细胞的文库构建。而且每个反应的试剂用量能够限制在纳升级，例如在本发明的一个实施例中，每个反应的试剂用量在300nL左右，这大大降低了实验成本。此外，转录组测序(RNA-seq)表明该方法构建的文库检测灵敏度高，重复性好，每个单细胞文库至少可以检测到10000个以上的基因。本发明的方法的开发成功，可以有效提高单细胞水平的RNA-seq建库速度，减少人工操作，节省高昂的试剂费用，实现单细胞建库的自动化和规模化。
在本发明中，控制细胞悬液的密度，对于高效率地将单细胞分配到微孔芯片的微孔中是比较关键的。发明人发现，在细胞均匀分布的情况下，当细胞密度小于等于4个/μL时，98％以上的微孔中细胞数是不多于1个。
在本发明的一个优选的实施例中，细胞密度为2-4个细胞/微升，如果细胞密度小于2个细胞/微升，可能有太多微孔中分配不到细胞，降低单细胞的有效分配率；当细胞密度大于4个细胞/微升，可能太多微孔中分配到不止一个细胞，也会相应地降低单细胞的有效分配率。
在本发明中，细胞悬液、细胞裂解液、反转录试剂和cDNA扩增试剂均是通过纳升级的微量分液平台分配到微孔中的，经过大量的实验研究发现，几十纳升级别的用量能够取得较好的效果，比如分配到微孔中的细胞悬液、细胞裂解液、反转录试剂和cDNA扩增试剂的体积可以均为35纳升至50纳升。
在本发明的一个优选的实施例中，采用Wafergen Biosystems公司的MSND作为微量分液平台，其最低分液体积为35纳升，因此在该优选的实施例中，分配到微孔中的细胞悬液、细胞裂解液、反转录试剂和cDNA扩增试剂的体积均为35纳升。同时，考虑到后续一些步骤的试剂用量，我们将微孔芯片的每个微孔的容积定制为350纳升。本领域技术人员可以理解，在一定范围内调整微孔的容积，定制不同规格的微孔芯片对实现本发明也是可行的，例如微孔的容积不小于350纳升均可以。我们分析，上述试剂的用量可以在几十纳升级别的用量范围内波动，然而太多或太少的试剂用量可能导致本发明的有效性降低，比 如超过100纳升的用量可能不但单细胞分离效果不好，随后的各步反应试剂用量也会增大，从而提高了成本。因此，本发明中，对于细胞悬液、细胞裂解液、反转录试剂和cDNA扩增试剂，不推荐100纳升以上的试剂用量。
由于本发明是对极微量的试剂进行分液操作，其中可能含有气泡，而气泡在极微量的试剂中可能难以除去，因此最好在将细胞悬液、反转录试剂或cDNA扩增试剂分配到微孔中后，通过离心去除气泡。如果cDNA扩增反应完成以后，还进行其它反应，也最好通过离心去除气泡。
本发明采用荧光定量PCR仪辅助监测单细胞扩增情况。如果以总RNA作为阳性对照，以细胞上清液作为阴性对照，典型的单细胞扩增产物的标志是，溶解曲线为单峰，Tm峰值大于等于88℃，同时Ct值介于阳性对照与阴性对照的数值之间。为了进一步提高单细胞选择的准确性，可以去除Ct值偏高或偏低的情况，例如去除最低和最高10％Ct值的样品。
虽然经过细胞裂解、反转录反应和cDNA扩增反应以后，已经实现了单个细胞的转录组文库的构建。但是，在实际应用中，通常还会进行后续反应。在本发明的一个优选的实施例中，在cDNA扩增反应之后：采用微量分液平台将TN5转座酶试剂分配到微孔中以对cDNA扩增反应的产物进行片段化处理，然后将终止反应试剂分配到微孔中以终止反应；之后，采用微量分液平台将PCR扩增反应试剂分配到微孔中以对片段化处理后的产物进行PCR文库扩增。
结合上述细胞悬液、细胞裂解液、反转录试剂和cDNA扩增试剂的用量情况，TN5转座酶试剂的用量可以为100纳升至150纳升；终止反应试剂的用量可以为35纳升至50纳升；PCR扩增反应试剂的用量可以为50纳升至75纳升。
在本发明的一个优选的实施例中，TN5转座酶试剂的用量为100纳升；终止反应试剂的用量为35纳升；PCR扩增反应试剂的用量为50纳升。
考虑到后续还可能进行混库(pooling)，即将不同单细胞的文库混合在一起。为了使混库之后的不同单细胞的转录组能够区别开来，可以在PCR文库扩增过程中，针对不同的微孔(即不同的单细胞)采用不同的引物对。在实际应用中，不同微孔中的两条引物只要其中一条序列不同即可。基于这样的思想，在本发明的一个优选实施方案中，引物对包括5’端引物和3’端引物，不同5’端引物之间的差别或不同3’端引物之间的差别仅在于各自具有一段专一性的标签序列，而其它部分的序列相同。具体地，标签序列可以是一段具有特定长度N(比如N为6-10的自然数，优选8)的随机序列。标签序列可以位于引物的各个位置， 优选位于引物的中段位置。
经过上述PCR文库扩增过程，不同的单细胞文库的两端具有了不同的引物序列。为了实现高通量测序等操作需求，可以将不同单细胞的文库混合在一起。因此，本发明的方法还可以包括，在PCR文库扩增之后：吸取根据荧光定量PCR仪辅助监测到的单细胞孔位中的产物并将其混合在一起。进一步，可以对混合在一起的产物，采用核酸纯化试剂盒纯化并选择预定长度的核酸片段。
以下通过实施例详细说明本发明的技术方案，需要说明的是，实施例用于说明本发明方法的可行性，不应当理解为对本发明保护范围的限制。
本实施例包括单细胞制备、单细胞裂解、反转录(RT)反应、cDNA扩增反应、TN5转座酶建库扩增等一系列步骤。试剂加样操作和PCR操作按照Wafergen Biosystems公司相关仪器说明进行操作。TN5转座酶按照Simone Picelli等(Genome Res.2014.24:2033-2040)发表的方法进行制备。
1.单细胞裂解液制备
配置如下裂解液(表1)，混合均匀，MSND平台进行加样，芯片中每孔加样35nL。2600rcf，12℃离心5min，去除气泡。
表1
裂解液组分 芯片孔用量(nL)
10％Triton X-100 0.5
40U/μl RNase抑制剂 1.25
10μM Oligo-dT30VN引物 12.5
10mm dNTP 12.5
ddH2O 8.25
总量 35
Oligo-dT30VN引物序列：
5′–AAGCAGTGGTATCAACGCAGAGTACT30VN-3′(SEQ ID NO：1)。
2.单细胞制备及裂解
2.1.使用Hela S3细胞系进行单细胞文库制备，使用胰酶对贴壁型Hela细胞进行消化，将消化掉的细胞悬浮液取1mL。将细胞在1000rpm条件下离心5min，用1×PBS重悬浮，重复一次。Percoll原液与10×PBS按体积比9:1混合成细胞的等渗溶液。将重悬的细胞加入配置的20％的等渗溶液中，细胞密度调节至4个/μL，完成细胞样品制备。
2.2.取10pg总RNA作为阳性对照，细胞悬浮液离心获得的上清液作为阴性对照。
2.3.采用MSND平台进行加样，芯片中每孔35nL体积。用膜封好，在2600rcf，12℃条件下离心5min，去气泡。
2.4.转移至可放置芯片的热循环仪中，72℃反应5min进行细胞裂解。裂解后将芯片在2600rcf，12℃条件下离心5min。
3.反转录反应
3.1.配置反转录试剂(表2)，混合均匀，MSND平台进行加样，芯片中每孔加样35nL。2600rcf，12℃离心5min，去除气泡。
表2
反转录试剂组分 芯片孔用量(nL)
200U/μL SSII 4.2
5×SuperScript II First-Strand Buffer 11.2
5M Betaine(甜菜碱) 11.2
100mM MgCl2 5.04
100mM DTT 1.4
100μM TSO 0.56
40U/μL RNAse抑制剂 1.4
总量 35
TSO序列：5′-AAGCAGTGGTATCAACGCAGAGTACATrGrG+G-3′(SEQ ID NO：2，rG表示riboguanosines，+G表示锁核酸修饰)。
3.2.转移芯片至热循环仪中，42℃90min；50℃2min,42℃2min，重复2次；72℃5min。反应后将芯片在2600rcf，12℃条件下离心5min。
4.cDNA扩增反应
4.1.配置cDNA扩增试剂(表3)，混合均匀，MSND平台进行加样，芯片中每孔加样35nL。2600rcf，12℃离心5min，去除气泡。加样完毕后，转移至荧光定量PCR仪中，进行PCR反应及溶解曲线测定。
表3
cDNA扩增试剂组分 芯片孔用量(nL)
2×KAPA HiFi HotStartReadyMix 29.19
10μM IS PCR引物 0.56
无核酸酶水 3.5
SYBR-Z(20×) 1.75
总量 35
IS PCR引物：5′-AAGCAGTGGTATCAACGCAGAGT-3′(SEQ ID NO：3)。
4.2.根据Ct值及溶解曲线判定单细胞扩增成功
选取溶解曲线为单峰，Tm峰值>＝88℃，同时Ct值介于阳性和阴性数值之间的样品。为了保证是单细胞扩增产物，去除最低和最高10％Ct值的样品。图1示出了部分样品的PCR反应及溶解曲线测定结果。
5.TN5转座酶建库
根据Simone Picelli等(Genome Res.2014.24:2033-2040)发表的方法进行制备，TN5酶浓度调至0.75mg/ml，配置5×打断缓冲液(50mM TAPS-NaOH，pH8.5(@RT)，25mM MgCl2，50％DMF)。
5.1接头(Adapter Mix)制备
5.1.1参考引物名称及序列：
引物A:5'-CTGTCTCTTATACACATCT-3'(SEQ ID NO：4)；
引物B:5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3′(SEQ ID NO：5)；
引物C:5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3′(SEQ ID NO：6)。
5.1.2使用退火缓冲液溶解引物A、引物B、引物C至100μM。
5.1.3分别配制如下反应体系，见表4：
表4
Figure PCTCN2015094777-appb-000001
5.1.4分别将反应1和反应2涡旋震荡充分混匀，并短暂离心使溶液回到管底。置于PCR仪内，进行如下反应程序：75℃15min；60℃10min；50℃10min；40℃10min；25℃30min。
5.1.5反应结束后，将反应1和反应2等体积混合，混匀。命名为接头(Adapter Mix)，-20℃保存。
5.2接头(Adapter Mix)包埋
5.2.1在灭菌PCR管中依次添加各反应组分，见表5：
表5
接头(Adapter Mix) 12.5μL
TN5 87.5μL
5.2.2使用移液器轻轻吹打，充分混匀。
5.2.3将反应置于25℃反应60min。反应产物命名为片段化酶高级混合物V5S(Tagment Enzyme Advanced Mix V5S)，置于-20℃存。
5.3DNA片段化
5.3.1配置TN5片段化试剂(表6)，混合均匀，MSND平台进行加样，芯片中每孔加样100nL。2600rcf，12℃离心5min，去除气泡。
表6
片段化试剂组分 芯片孔用量(nL)
5×缓冲液 45
Tagment Enzyme Advanced Mix V5S 55
总量 100
5.3.2 55℃热循环仪中温浴7min。
5.4终止反应
配置打断终止反应试剂(表7)，其中384孔板中每孔添加一种N5XX引物，混合均匀，MSND平台选择(72×72模式)加样，芯片中每孔加样35nL，2600rcf，25℃离心5min，去除气泡。室温放置5min。
表7
打断终止反应试剂组分 芯片孔用量(nL)
2.25％SDS 1.45
N5XX(0.5μM) 6.5
引物1(10μM) 6.5
引物2(10μM) 6.5
5×KAPA Fidelity Buffer 14.05
总量 35
引物N5XX序列：
AATGATACGGCGACCACCGAGATCTACACXXXXXXXXTCGTCGGCAG CGTC(SEQ ID NO：7，X表示随机碱基)；
引物1序列：5′-AATGATACGGCGACCACCGA-3′(SEQ ID NO：8)；
引物2序列：5′-CAAGCAGAAGACGGCATACGA-3′(SEQ ID NO：9)。
5.5PCR文库扩增
配置扩增反应试剂(表8)，384孔板中每孔添加一种N7XX引物混合均匀，MSND平台选择(72×72模式)加样，芯片中每孔加样50nL，2600rcf，12℃离心5min，去除气泡。
表8
Figure PCTCN2015094777-appb-000002
引物N7XX序列：
CAAGCAGAAGACGGCATACGAGATXXXXXXXXGTCTCGTGGGCTCGG(SEQ ID NO：10，X表示随机碱基)。
将芯片放入热循环仪中进行扩增。
设置如下反应程序：
热盖105℃；72℃3min；95℃3min；(98℃20sec；60℃15sec；72℃25sec)×15-20循环；72℃5min；4℃保温。
6.根据4.2，将判定单细胞成功扩增出来的孔位，使用直径小于200μm的玻璃针或者特制的吸量仪吸取混库(pooling)在一起。
7.将混库后的产物使用Agencourt AMPure XP beads，进行选择性纯化。
7.1涡旋震荡混匀AMPure XP beads并吸取40μL体积至50μL PCR混库后的产物中，使用移液器轻轻吹打10次充分混匀。室温孵育5分钟。
7.2将反应管短暂离心并置于磁力架中分离磁珠和液体。待溶液澄清(约5分钟)小心转移上清至干净EP管中，丢弃磁珠。
7.3涡旋震荡混匀AMPure XP beads并吸取7.5μL体积至上清中，使用移液器轻轻吹打10次充分混匀。室温孵育5分钟。
7.4将反应管短暂离心并置于磁力架中分离磁珠和液体。待溶液澄清(约5分钟)小心移除上清。
7.5保持EP管始终处于磁力架中，加入200μL新鲜配制的80％乙醇漂洗磁珠。室温孵育30秒后小心移除上清。
7.6重复上步，总计漂洗两次。
7.7保持EP管始终处于磁力架中，开盖空气干燥磁珠10分钟。
7.8将EP管从磁力架中取出，加入15μL灭菌超纯水洗脱。涡旋振荡或使用移液器轻轻吹打充分混匀。将反应管短暂离心并置于磁力架中分离磁珠和液体。待溶液澄清(约5分钟)小心吸取上清至灭菌EP管中，于-20℃保存。如需获得长度分布更集中的文库，扩增产物可使用胶回收试剂盒进行片段长度分选和纯化。
8Agilent 2100检测文库构建结果。结果如图2。
9根据纯化结果选择不同测序类型进行illumina上机测序。测序结果如图3。
以上内容是结合具体的实施方式对本发明所作的进一步详细说明，不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干简单推演或替换，都应当视为属于本发明的保护范围。

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  1. 一种高通量的单细胞转录组建库方法，其特征在于，所述方法包括：
    采用纳升级的微量分液平台将具有预定细胞密度的细胞悬液分配到纳升级的微孔芯片的每个微孔中，用细胞裂解液裂解所述微孔内的细胞；
    采用所述微量分液平台将反转录试剂分配到所述微孔中进行反转录反应；
    采用所述微量分液平台将cDNA扩增试剂分配到所述微孔中进行cDNA扩增反应，同时采用荧光定量PCR仪辅助监测单细胞扩增情况。
  2. 根据权利要求1所述的方法，其特征在于，所述预定细胞密度是指2-4个细胞/微升。
  3. 根据权利要求1所述的方法，其特征在于，分配到所述微孔中的细胞悬液、细胞裂解液、反转录试剂和cDNA扩增试剂的体积均为35纳升至50纳升。
  4. 根据权利要求1所述的方法，其特征在于，分配到所述微孔中的细胞悬液、细胞裂解液、反转录试剂和cDNA扩增试剂的体积均为35纳升。
  5. 根据权利要求4所述的方法，其特征在于，所述微孔芯片的每个微孔的容积为不小于350纳升。
  6. 根据权利要求1所述的方法，其特征在于，将所述细胞悬液、反转录试剂或cDNA扩增试剂分配到所述微孔中后，通过离心去除气泡。
  7. 根据权利要求1所述的方法，其特征在于，所述荧光定量PCR仪辅助监测单细胞扩增情况中，以溶解曲线为单峰，Tm峰值大于等于88℃，同时Ct值介于阳性对照与阴性对照的数值之间，作为单细胞扩增产物的标志；其中所述阳性对照是总RNA，所述阴性对照是细胞上清液。
  8. 根据权利要求7所述的方法，其特征在于，所述方法还包括：去除最低和最高10％Ct值的样品。
  9. 根据权利要求1所述的方法，其特征在于，所述方法还包括，在所述cDNA扩增反应之后：
    采用所述微量分液平台将TN5转座酶试剂分配到所述微孔中以对所述cDNA扩增反应的产物进行片段化处理，然后将终止反应试剂分配到所述微孔中以终止反应；
    采用所述微量分液平台将PCR扩增反应试剂分配到所述微孔中以对所述片段化处理后的产物进行PCR文库扩增。
  10. 根据权利要求9所述的方法，其特征在于，所述TN5转座酶试剂的体积为100纳升至150纳升；所述终止反应试剂的体积为35纳升至50纳升；所 述PCR扩增反应试剂的体积为50纳升至75纳升。
  11. 根据权利要求9所述的方法，其特征在于，所述TN5转座酶试剂的体积为100纳升；所述终止反应试剂的体积为35纳升；所述PCR扩增反应试剂的体积为50纳升。
  12. 根据权利要求9所述的方法，其特征在于，所述PCR文库扩增过程中，不同的微孔中采用不同的引物对。
  13. 根据权利要求12所述的方法，其特征在于，所述引物对包括5’端引物和3’端引物，不同5’端引物之间的差别或不同3’端引物之间的差别仅在于各自具有一段专一性的标签序列，而其它部分的序列相同。
  14. 根据权利要求9所述的方法，其特征在于，所述方法还包括，在所述PCR文库扩增之后：
    吸取根据所述荧光定量PCR仪辅助监测到的单细胞孔位中的产物并将其混合在一起；
    对所述混合在一起的产物，采用核酸纯化试剂盒纯化并选择预定长度的核酸片段。
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