WO2022262102A1 - 一种用于单细胞测序的牛瘤胃上皮组织解离方法 - Google Patents

一种用于单细胞测序的牛瘤胃上皮组织解离方法 Download PDF

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WO2022262102A1
WO2022262102A1 PCT/CN2021/112671 CN2021112671W WO2022262102A1 WO 2022262102 A1 WO2022262102 A1 WO 2022262102A1 CN 2021112671 W CN2021112671 W CN 2021112671W WO 2022262102 A1 WO2022262102 A1 WO 2022262102A1
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centrifugation
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孙会增
吴家劲
刘潇菡
朱森林
顾凤飞
刘建新
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Zhejiang University
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0679—Cells of the gastro-intestinal tract
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    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
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    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
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    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
    • C12N2509/10—Mechanical dissociation

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  • the invention relates to the field of cell biology, in particular to a bovine rumen epithelial tissue dissociation method for single-cell sequencing.
  • the rumen is a unique digestive organ of ruminants such as cows, which plays a vital role in the digestion, metabolism, absorption and transport of nutrients.
  • Epithelial tissue is the main place where the rumen functions, but how to maintain and improve its structure and function in beef and dairy cattle farming still faces severe challenges, such as subacute rumen acidosis. Therefore, in-depth analysis of the key physiological functions and regulatory mechanisms of the rumen of dairy cows has become an important way to improve the health and production performance of dairy cows.
  • Cells are the basic unit of life activities.
  • the rumen epithelium is located on the innermost side of the rumen wall. It is a stratified squamous epithelium with four layers of multifunctional cell layer structure. The protruding layer, granular layer and stratum corneum, but the study of their cell types and molecular characteristics is still obviously insufficient.
  • the purified cell suspension contains various types of cells such as basal layer, spinous process layer and even granular layer cells, so the existing research is based on a large class of epithelial cells and cannot be in-depth This also leads to the fact that the function of dairy cow rumen epithelial tissue cannot be accurately analyzed at the cellular level; at the same time, the interaction between different types of cells in rumen epithelial tissue, the function of a small number of mesenchymal cells, A series of basic and important issues such as the trajectory of cell differentiation and development.
  • the invention successfully establishes for the first time the technology of rumen epithelial tissue dissociation and single cell suspension preparation for single cell sequencing such as single cell transcriptome, which will promote the development of rumen single cell research in ruminants. At the same time, it will establish a good foundation for other single-cell-based research in the future, such as single-cell epigenetics and single-cell proteomics.
  • the purpose of the present invention is to address the deficiencies of the prior art, to propose a bovine rumen epithelial tissue dissociation method for single-cell sequencing, to obtain a single-cell suspension of rumen tissue with high activity, high cell number, and low pollution, and to ensure effective single-cell
  • the output of data and the quality of single-cell nucleic acid are convenient for single-cell omics sequencing and data analysis.
  • a bovine rumen epithelial tissue dissociation method for single-cell sequencing specifically comprising the following steps:
  • step (1) Add trypsin/EDTA to the last cut tissue piece in step (1) for digestion; after digestion, insert into ice and add HBSS to stop digestion; after centrifugation, wash again with HBSS; add the incubated enzyme dissociation solution, and the enzyme dissociation
  • the solution contains neutral protease, collagenase I, collagenase IV, hyaluronidase and DNase I, after incubation, centrifuge; then add FBS to inhibit enzyme activity, and obtain a digested cell suspension;
  • step (2) Filter the digested cell suspension in step (2) with a cell strainer and collect it into a new EP tube; discard the supernatant after centrifugation;
  • step (4) Use a cell strainer to filter the cell suspension obtained in step (4), centrifuge after washing the filter membrane with PBS containing BSA; observe the cell debris, if the debris rate is greater than 10%, repeat this step;
  • step (5) Blow evenly the cell suspension obtained in step (5), take part of the cell suspension and place it in a hemocytometer, observe the background debris, cell clumping and cell density with a microscope; add trypan blue staining, calculate cell viability, cell concentration and total cells. If it is observed that the cell activity does not meet the requirements for cell activity of single-cell sequencing, but the total number of cells is sufficient, the dead cell removal operation is required.
  • a dead cell removal device can be used to remove dead cells.
  • the specific operation is as follows: In the ultra-clean bench, dilute 20 ⁇ Binding Buffer with double distilled water to 1 ⁇ Binding Buffer; centrifuge the cell suspension obtained in step (5) and discard the supernatant; collect the cell pellet, add Dead Cell Removal MicroBeads were blown and mixed; incubated at room temperature, during which the diluted Binding Buffer was added; after the incubation, before the washing solution exposed the column, the diluted Binding Buffer was added to the cell suspension and added to the top of the column; washed with the diluted Binding Buffer Column; centrifuge the collection tube and discard the supernatant; add BSA-containing PBS to resuspend the cells; mix by pipetting and continue to step (6) for observation.
  • the tissue block is first chopped into small pieces with a volume of 10 ⁇ 0.5 mm 2 , and when the tissue is further chopped, the tissue block is chopped until the volume of the tissue block is 1 mm 3 .
  • the PBS is 1 ⁇ PBS, and the amount added is 5 ml; the DPBS is 1 ⁇ DPBS.
  • the ratio of the volume of EDTA added to the volume of the tissue block is 2:1.
  • step (1) the EDTA concentration is 20 mM, and soaked at 37° C. for 30 min.
  • the concentration of the trypsin/EDTA is 2500mg/L, and the ratio of the added volume to the tissue block after the tissue is further shredded is 2:1;
  • the digestion conditions are: digestion temperature The temperature is 37°C, and the digestion time is 5 minutes.
  • the cooling time in ice is 2 min.
  • the volume of the HBSS is equal to that of the cell suspension, and the HBSS is pre-cooled HBSS;
  • the centrifugation conditions are: the centrifugation speed is 300 ⁇ g, the centrifugation temperature is 4° C., and the centrifugation time is 2 minutes.
  • the ratio of the volume of the enzyme dissociation solution added to the tissue block after the tissue is continued to be shredded is 2:1, which contains 1.5mg/ml neutral protease, 1.5mg/ml collagenase I, 1.5mg/ml collagenase IV, 100U/ml hyaluronidase, 50U/ml DNase I; the reaction conditions are: the incubation temperature is 37°C, and the incubation time is 30min.
  • the FBS is 10% FBS (V/V), and the volume is the same as that of the enzyme dissociation solution added.
  • step (3) the filtration uses stacked cell filters with pore sizes of 30 ⁇ m and 70 ⁇ m.
  • the centrifugation conditions are as follows: the centrifugation speed is 300 ⁇ g, the centrifugation temperature is 4° C., and the centrifugation time is 5 minutes.
  • the amount of HBSS added is 2 mL.
  • the amount of PBS added was 2 mL; the PBS was 1 ⁇ PBS, and the PBS contained BSA at a concentration of 400 mg/L.
  • the centrifugation conditions are as follows: the centrifugation speed is 300 g, the centrifugation temperature is 4° C., and the centrifugation time is 5 min.
  • the cell filter uses a Miltenyi 20 ⁇ m cell filter; the PBS is 1 ⁇ PBS, and the PBS contains BSA at a concentration of 400 mg/L.
  • the centrifugation conditions are as follows: the centrifugation speed is 300 g, the centrifugation temperature is 4° C., and the centrifugation time is 5 min.
  • the device for removing dead cells includes MS or LS columns, magnets, racks, and receiving tubes.
  • MS or LS the amount of Binding Buffer used is different.
  • the diluted Binding Buffer is 1 ⁇ Binding Buffer; the usage condition of MS is 3-4ml/rxn, and the usage condition of LS is 17-26ml/rxn.
  • the amount of Binding Buffer added during incubation is: add 500 ⁇ l to MS, add 3ml to LS; after incubation, the amount of Binding Buffer added is: add 500-1000 ⁇ l to MS; add 1-10ml to LS.
  • the amount of Binding Buffer added is: 500 ⁇ l each time in MS, 4 times in total; 3ml each time in LS, 4 times in total.
  • the amount of Dead Cell Removal MicroBeads added is 100 ⁇ l.
  • the incubation time is 15 minutes, and the time of adding Binding Buffer is the 14th minute of incubation; the centrifugation conditions are: the centrifugation speed is 300 ⁇ g, the centrifugation temperature is 4°C, and the centrifugation time is 5-10 minutes.
  • the amount of PBS added is 50-300 ⁇ l; the PBS is 1 ⁇ PBS, and the PBS contains BSA at a concentration of 400 mg/L.
  • the beneficial effects of the present invention are as follows: there is no specific and systematic single-cell dissociation method for bovine rumen tissue, and the present invention achieves a new breakthrough.
  • the invention can obtain single-cell suspension of rumen tissue with high activity, high cell number, low pollution and complete cell types, ensuring the output of effective single-cell number and the quality of single-cell mRNA, so as to facilitate single-cell sequencing and data analysis.
  • Fig. 1 is the image under the microscope of bovine rumen tissue single-cell suspension that parallel experiment (1) obtains in the specific embodiment: background debris is less, cell clumping situation almost does not have, and cell density is moderate;
  • Fig. 2 is the image under the microscope of bovine rumen tissue single-cell suspension that parallel experiment (2) obtains in the specific embodiment: background debris is less, the cell clumping situation hardly has, and cell density is moderate;
  • Fig. 3 is the image under the microscope of bovine rumen tissue single-cell suspension obtained in parallel experiment (3) in the specific embodiment: background fragments are few, cell agglomeration situation almost does not have, and cell density is moderate;
  • Fig. 4 is the cell concentration of the bovine rumen tissue single cell suspension that parallel experiment (1) in the specific embodiment, (2) and (3) obtains, and sample one, two, three are used in Fig. 1,2,3 respectively The samples are the same: the cell concentrations are 1.15 ⁇ 10 6 /mL, 1.27 ⁇ 10 6 /mL and 2.64 ⁇ 10 6 /mL, exceeding 10 6 /mL;
  • Fig. 5 is the cell viability rate of the bovine rumen tissue single cell suspension that parallel experiment (1) in the specific embodiment, (2) and (3) obtains, and sample one, two, three are respectively with Fig. 1,2,3. The same sample was used: the cell viability was 88%, 94% and 94% respectively, reaching the standard of 85%;
  • Fig. 6 is the cDNA quality inspection figure of the bovine rumen tissue single-cell suspension obtained in the specific embodiment: the main peak of the fragment distribution figure is greater than 1000bp and there is no jagged miscellaneous peak, and it is judged as qualified;
  • Fig. 7 is the library quality inspection picture of bovine rumen tissue single-cell suspension obtained in the specific embodiment: according to the requirements of the sequencing platform, the size of the library is generally about 300-600bp, including sequencing joints, read1, read2, and 10X barcode , UMI, Poly DT, insert fragment and sample index; in a specific embodiment, it is judged qualified in the case of the main peak 450bp.
  • Fig. 8 is a flowchart of the method of the present invention.
  • a bovine rumen epithelial tissue dissociation method for single-cell sequencing provided by the present invention is shown in Figure 8, and the specific steps are:
  • Tissue enzymatic hydrolysis Add 5ml 0.25% trypsin/EDTA to the EP tube containing the 1mm3 tissue block in step (1), digest at 37°C for 5min; insert it into ice for 2min quickly after digestion, then add Volume pre-cooled HBSS, stop digestion; then centrifuge at 4°C, 300 ⁇ g for 2min, then add 2ml HBSS, centrifuge at 300 ⁇ g for 2min, wash again; then add 2ml of incubated enzyme dissociation solution (1.5mg/ ml neutral protease, 1.5mg/ml collagenase I, 1.5mg/ml collagenase IV, 100U/ml hyaluronidase, 50U/ml DNase I), incubate at 37°C for 30min; then directly add 2ml 10% FBS (V /V), inhibit enzyme activity;
  • step (6) Cell observation and quality inspection: Gently blow the cell suspension obtained in step (5), take about 6 ⁇ L and place it in a hemocytometer, observe the background debris, cell clumping and cell density with a microscope; add trypan blue Staining, calculating cell viability, cell concentration and cell number;
  • Dead cell removal If the cell activity observed in step (6) does not meet the cell activity requirements of single-cell sequencing, but the total amount of cells is sufficient, the dead cell removal device can be used to remove dead cells.
  • step (1) and step (2) are crucial to the high dissociation efficiency of the present invention.
  • the present invention also provides a high-efficiency method for the dissociation of bovine rumen tissue samples.
  • the method includes that Ca 2+ and Mg 2+ are required to maintain the integrity of the tissue due to the tightness of the bovine rumen epithelial tissue, and EDTA can Absorb these ions from the living environment of these tissues, form chelates, promote the separation of cells, make the tissues soft, and make the enzyme dissociation solution added later act better on the cells.
  • the present invention also provides a tissue enzyme dissociation solution for single-cell dissociation of bovine rumen tissue samples.
  • ml collagenase I (Worthington, A004214-0050), 1.5mg/ml collagenase IV (Worthington, A004210-0050), 100U/ml hyaluronidase (Worthington, A005477-0005), 50U/ml DNase I (Sigma, 11284932001).
  • the present invention also provides the application of the tissue enzyme dissociation solution in dissociation of bovine rumen tissue samples.
  • An example of the present invention is as follows:
  • the quality inspection of cDNA and library was carried out. As shown in Figure 6 and Figure 7, they are the cDNA quality inspection map and the library quality inspection map of the single-cell suspension of bovine rumen tissue respectively.
  • the main peak of the cDNA quality inspection picture segment distribution map is greater than 1000bp and there is no jagged peak, which is judged as Passed; according to the requirements of the sequencing platform, the library size is generally around 300-600bp, including sequencing adapters, read1, read2, 10X barcode, UMI, Poly DT, insert fragments, and sample index; it is judged qualified when the main peak is 450bp.

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Abstract

提供了一种用于单细胞测序的牛瘤胃上皮组织解离方法，包括瘤胃组织机械分离、酶消化处理、杂质过滤、细胞清洗、再次过滤、细胞观察和质控、死细胞去除等。该方法可以获得高活性、高细胞数、低污染的瘤胃组织单细胞悬液，保证有效单细胞数的产出和单细胞核酸的质量，解决现有技术中尚无系统完善的牛瘤胃单细胞解离技术的问题。

Description

一种用于单细胞测序的牛瘤胃上皮组织解离方法 技术领域
本发明涉及细胞生物学领域，尤其涉及一种用于单细胞测序的牛瘤胃上皮组织解离方法。
背景技术
瘤胃是奶牛等反刍动物特有的消化器官，在营养物质消化代谢和吸收转运中起着至关重要的作用。上皮组织是瘤胃行使功能的主要场所，而在肉牛和奶牛养殖中如何维持和改善其结构功能仍面临着严峻挑战，如亚急性瘤胃酸中毒等。因此，深入解析奶牛瘤胃关键生理功能和调节机制成为改善奶牛健康和生产性能的重要途径。细胞是生命活动的基本单元，瘤胃上皮组织位于瘤胃壁的最内侧，是一种复层鳞状上皮，具有四层多功能细胞层结构，即从最外到瘤胃腔内依次为基底层、棘突层、颗粒层和角质层，但其细胞类型及分子特征的研究还明显不足。
目前奶牛瘤胃上皮组织功能的研究多在组织水平或体外培养的瘤胃上皮细胞。通过对瘤胃上皮组织进行转录组分析，我们捕获到特定条件下所有的表达基因，进而解析了其在不同外界处理或刺激下系统功能的变化。然而1g瘤胃上皮组织中至少包含上百万个细胞，传统组织水平的转录组分析忽略了细胞异质性，无法获得细胞水平的功能特征。基于细胞水平的方法中，纯化后的细胞悬液中包含了多种类型的细胞如基底层、棘突层甚至颗粒层细胞，因此现有研究都是建立在大类的上皮细胞上而无法深入到单一细胞类型进行探究，这也导致了奶牛瘤胃上皮组织的功能仍然不能在细胞水平精确地予以解析；同时也无法解答瘤胃上皮组织不同类型细胞之间的互作、少量间质细胞的功能、细胞分化发育轨迹等一系列基础而重要的问题。
单细胞水平研究的一个重要前提，是建立符合单细胞捕获追踪条件的组织充分解离和单细胞悬液制备方法；与瘤胃上皮细胞分离培养方法相比的最大区别在于，该类方法需要尽可能充分解离组织以获得所有类型细胞，并能将这些细胞高效率捕获并特异性地得以追踪。我们前期研究也发现利用传统瘤胃上皮细胞体外分离培养方法获得的细胞悬液无法满足单细胞测序的要求，需要在方法上进一步探索。本发明首次成功建立了用于单细胞转录组等单细胞测序的瘤胃上皮组织解离和单细胞悬液制备技术，将推动开启反刍动物瘤胃单细胞研究。同时，为将来开展其他以单细胞为基础的研究如单细胞表观组、单细胞蛋白质组等建立良好基础。
发明内容
本发明目的在于针对现有技术的不足，提出一种用于单细胞测序的牛瘤胃上皮组织解离方法，获得高活性、高细胞数、低污染的瘤胃组织单细胞悬液，保证有效单细胞数的产出和单细胞核酸的质量，以便于单细胞组学测序和数据分析。
本发明的目的是通过以下技术方案来实现的：一种用于单细胞测序的牛瘤胃上皮组织解离方法，具体包括以下步骤：
(1)瘤胃组织的机械分离
将无菌无RNA酶的细胞培养皿置于冰盒上，加入PBS；将瘤胃组织置于培养皿中，DPBS清洗后，使用灭菌手术剪刀镊子去除肌肉层，将组织块剪碎，放入EDTA浸泡，之后再次使用DPBS清洗，将组织继续剪碎，弃上清；
(2)酶消化处理
向步骤(1)最后剪碎的组织块中加入胰酶/EDTA消化；消化后插入冰里加入HBSS停止消化；离心后用HBSS再次清洗；加入孵育好的酶解离溶液，所述酶解离溶液包含中性蛋白酶、胶原酶I、胶原酶IV、透明质酸酶和DNase I，孵育结束后再离心；之后加入FBS抑制酶活性，得到消化好的细胞悬液；
(3)杂质过滤
用细胞过滤器过滤步骤(2)消化好的细胞悬液，收集至新的EP管；离心后弃上清；
(4)细胞清洗
向步骤(3)离心后的细胞沉淀中加入HBSS重悬，离心后弃上清；加入含有BSA的PBS进行重悬，离心后弃上清；再次加入含有BSA的PBS重悬细胞，得到细胞悬液；
(5)再次过滤
使用细胞过滤器过滤步骤(4)得到的细胞悬液，使用含有BSA的PBS冲洗滤膜后离心；观察细胞碎片情况，如果碎片率大于10％，重复该步骤；
(6)细胞观察和质检
吹匀步骤(5)得到的细胞悬液，取部分细胞悬液置于血球计数板中，用显微镜观察背景碎片、细胞结团和细胞密度；加入台盼蓝染色，计算细胞活性、细胞浓度和细胞总量。如果观察到细胞活性没有满足单细胞测序对细胞活性的要求，但细胞总量足量时需要进行死细胞去除操作。
进一步地，如果步骤(6)中观察到细胞活性没有满足单细胞测序对细胞活性的要求，但细胞总量足量时可使用去死细胞装置进行死细胞去除。具体操作如下：在超净台内，将20×Binding Buffer用双蒸水稀释成1×Binding Buffer；将步骤(5)得到的细胞悬液离心后弃上清；收集细胞沉淀，加入Dead Cell Removal MicroBeads吹打混匀；室温孵育，期间加入稀释 后的Binding Buffer；孵育结束之后，润洗液暴露柱子之前，向细胞悬液中加入稀释后的Binding Buffer并加入柱子上面；使用稀释后的Binding Buffer冲洗柱子；将收集管离心后弃上清；加入含BSA的PBS重悬细胞；吹打混匀，继续步骤(6)观察。
进一步地，步骤(1)中，首先将组织块剪碎成小块，体积为10×0.5mm 2，将组织继续剪碎时，剪碎至组织块体积为1mm 3。所述PBS为1×PBS，加入量为5ml；所述DPBS为1×DPBS。所述EDTA加入的体积与组织块体积之比为2：1。
进一步地，步骤(1)中，所述EDTA浓度为20mM，在37℃温度条件下浸泡30min。
进一步地，步骤(2)中，所述胰酶/EDTA的浓度为2500mg/L，加入的体积与将组织继续剪碎后的组织块之比为2：1；所述消化条件为：消化温度为37℃，消化时间为5min。冰里冷却时间为2min。所述HBSS体积为与细胞悬液相等体积，所述HBSS为预冷HBSS；所述离心条件为：离心转速为300×g，离心温度为4℃，离心时间为2min。所述酶解离溶液加入的体积与将组织继续剪碎后的组织块之比为2：1，其包含1.5mg/ml中性蛋白酶、1.5mg/ml胶原酶I、1.5mg/ml胶原酶IV、100U/ml透明质酸酶、50U/mlDNase I；反应条件为：孵育温度为37℃，孵育时间为30min。所述FBS为10％FBS(V/V)，体积与加入酶解离溶液体积相同。
进一步地，步骤(3)中，所述过滤使用30μm和70μm孔径的堆叠的细胞过滤器。所述离心条件为：离心转速为300×g，离心温度为4℃，离心时间为5min。
进一步地，步骤(4)中，所述HBSS的加入量为2mL。所述PBS的加入量为2mL；所述PBS为1×PBS，所述PBS含有浓度为400mg/L的BSA。所述离心条件均为：离心的转速为300g，离心的温度为4℃，离心的时间为5min。
进一步地，所述步骤(5)中，所述细胞过滤器使用美天旎20μm细胞过滤器；所述PBS为1×PBS，所述PBS含有浓度为400mg/L的BSA。所述离心条件均为：离心的转速为300g，离心的温度为4℃，离心的时间为5min。
进一步地，所述去死细胞装置包括MS或LS柱子、磁铁、架子、接收管。对于MS或LS，Binding Buffer使用量不同，所述稀释后的Binding Buffer为1×Binding Buffer；MS的使用条件为3-4ml/rxn，LS的使用条件为17-26ml/rxn。孵育时Binding Buffer加入量为：MS中加入500μl，LS中加入3ml；孵育结束后，Binding Buffer的加入量为：MS中加入500-1000μl；LS中加入1-10ml。冲洗柱子时，Binding Buffer的加入量为：MS中每次500μl，共4次；LS中每次3ml，共4次。
进一步地，所述Dead Cell Removal MicroBeads的添加量为100μl。所述孵育的时长为15min，加入Binding Buffer的时间为孵育的第14min；所述离心条件均为：离心的转速为300 ×g，离心的温度为4℃，离心的时间为5-10min。所述PBS的加入量为50-300μl；所述PBS为1×PBS，所述PBS含有浓度为400mg/L的BSA。
与现有技术相比，本发明的有益效果如下：现今并未有具体的、系统的针对牛瘤胃组织的单细胞解离方法，本发明实现了一个新的突破。本发明可获得高活性、高细胞数、低污染的、细胞类型齐全瘤胃组织单细胞悬液，保证有效单细胞数的产出和单细胞mRNA的质量，以便于单细胞测序及数据分析。
附图说明
图1是具体实施方式中平行实验(1)得到的牛瘤胃组织单细胞悬液在显微镜下的图像：背景碎片较少，细胞结团情况几乎没有，细胞密度适中；
图2是具体实施方式中平行实验(2)得到的牛瘤胃组织单细胞悬液在显微镜下的图像：背景碎片较少，细胞结团情况几乎没有，细胞密度适中；
图3是具体实施方式中平行实验(3)得到的牛瘤胃组织单细胞悬液在显微镜下的图像：背景碎片较少，细胞结团情况几乎没有，细胞密度适中；
图4是具体实施方式中平行实验(1)，(2)和(3)得到的牛瘤胃组织单细胞悬液的细胞浓度，样本一、二、三分别与图1、2、3中所使用样本相同：细胞浓度分别为1.15×10 6/mL，1.27×10 6/mL和2.64×10 6/mL，超过10 6/mL；
图5是具体实施方式中平行实验(1)，(2)和(3)得到的牛瘤胃组织单细胞悬液的细胞活率，样本一、二、三分别与图1、2、3中所使用样本相同：细胞活率为分别为88％，94％和94％，达到85％的标准；
图6是具体实施方式中所得到的牛瘤胃组织单细胞悬液的cDNA质检图：片段分布图的主峰值大于1000bp且无锯齿状杂峰，判定为及格；
图7是具体实施方式中所得到的牛瘤胃组织单细胞悬液的library质检图：根据测序平台的要求，文库大小一般在300-600bp左右，其中包括了测序接头、read1、read2、10X barcode、UMI、Poly DT，插入片段以及样本index；在具体的实施方式中，在主峰450bp的情况下判定合格。
图8为本发明方法流程图。
具体实施方式
下面结合具体实施例对本发明进行详细说明。以下实施例将有助于本领域的技术人员进一步理解本发明，但不以任何形式限制本发明。应当指出的是，对本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进。这些都属于本发明的保护范围。
本发明提供的一种用于单细胞测序的牛瘤胃上皮组织解离方法，如图8所示，具体步骤为：
(1)样本机械处理：将无菌无RNA酶细胞培养皿置于冰盒上，加入1×PBS；挑取牛瘤胃组织置于培养皿上，使用1×DPBS清洗2遍，观察肌层残留情况，使用剪刀镊子去除肌层，然后将组织块剪碎为10×0.5mm 2小块，转移至EP管中，加入组织体积2倍量的20mM EDTA于37℃浸泡30min；接着使用1×DPBS清洗2遍，将组织继续剪碎至1mm 3，弃上清，挑取适量组织块于新的EP管中；
(2)组织酶解：向步骤(1)中含有1mm 3组织块的EP管中加入5ml 0.25％胰酶/EDTA，在37℃条件下消化5min；消化后迅速插入冰里2min，然后加入等体积预冷的HBSS，停止消化；接着在4℃，300×g条件下离心2min，之后加入2ml HBSS，300×g离心2min，再次清洗；然后加入孵育好的2ml酶解离溶液(1.5mg/ml中性蛋白酶、1.5mg/ml胶原酶I、1.5mg/ml胶原酶IV、100U/ml透明质酸酶、50U/mlDNase I)，37℃孵育30min；随后直接加入2ml 10％的FBS(V/V)，抑制酶活性；
(3)过滤：用30和70μm孔径的堆叠细胞过滤器过滤步骤(2)消化好的细胞悬液，收集过滤好的细胞悬液于新的EP管，300×g，4℃条件下离心5min，弃上清。
(4)细胞清洗：向步骤(3)中含有细胞沉淀的EP管中加入2mlHBSS重悬细胞以清洗细胞，在300×g，4℃条件下离心5min，弃上清；再向EP管中加入2ml 1×PBS(含0.04％BSA，即浓度为400mg/L的BSA)重悬细胞以清洗细胞，在300×g，4℃条件下离心5min，弃上清；然后再加入2ml 1×PBS(含0.04％BSA)重悬细胞。
(5)再次过滤：使用美天旎20μm细胞过滤器过滤步骤(4)中消化好的细胞悬液，加入2ml 1×PBS(含0.04％BSA)冲洗滤膜，然后在300×g，4℃条件下离心5min；观察细胞碎片情况，如果碎片率>10％，重复该步骤；
(6)细胞观察和质检：轻缓吹匀步骤(5)得到的细胞悬液，取6μL左右置于血球计数板中，用显微镜观察背景碎片、细胞结团和细胞密度；加入台盼蓝染色，计算细胞活率、细胞浓度和细胞数目；
(7)死细胞去除(选做)：如果步骤(6)中观察到细胞活性没有满足单细胞测序对细胞活性的要求，但细胞总量足量时可使用去死细胞装置进行死细胞去除。首先将专用的MS或者LS柱子、磁铁、架子、接收管放置在超净台上面准备去除死细胞；在超净台内，使用双蒸水将20×Binding Buffer稀释成1×Binding Buffer(MS：3-4ml/rxn或者LS：17-26ml/rxn)；300g，4℃条件下离心，弃上清；收集细胞沉淀，加入100μL Dead Cell Removal MicroBeads吹打混匀，室温孵育15min；在孵育时间14min左右，加入1×Binding Buffer(MS:500μl；LS: 3ml)；孵育结束之后，润洗液暴露柱子之前，向细胞悬液中加入1×Binding Buffer(MS:500-1000μl；LS:1-10ml)，并加入柱子上面；使用1×Binding Buffer(MS:4×500μl；LS:4×3ml)冲洗柱子；将收集管在300×g，4℃条件下离心，弃上清；然后加入合适体积的50-300μmL(根据细胞沉淀的多少判断加入的量)的0.04％BSA 1×PBS重悬细胞；使用1ml移液枪吹打混匀5次，继续步骤(6)观察，直至细胞总量和细胞活性均达到要求。
本发明以上牛瘤胃组织解离步骤中，步骤(1)和步骤(2)对本发明获得高效解离效率是至关重要的。
本发明还提供了一种用于牛瘤胃组织样本解离的高效方法，所述方法包括由于牛瘤胃上皮组织紧密，在生存中需要Ca 2+和Mg 2+才能维持组织的完整性，EDTA能从这些组织生存环境中吸收这些离子，形成螯合物，促进细胞相互分离，使组织松软，使后面加入的酶解离液更好地作用于细胞。
本发明还提供了一种用于牛瘤胃组织样本单细胞解离的组织酶解离溶液，所述组织酶解离溶液包括1.5mg/ml中性蛋白酶(Worthington，A02100-0010)、1.5mg/ml胶原酶I(Worthington，A004214-0050)、1.5mg/ml胶原酶IV(Worthington，A004210-0050)、100U/ml透明质酸酶(Worthington，A005477-0005)、50U/ml DNase I(Sigma，11284932001)。
本发明还提供了所述组织酶解离液在用于解离牛瘤胃组织样本中的应用。
本发明的一个实施例如下：
实验前准备
(1)在实验前，无菌操作台灭菌至少30分钟；
(2)称量好特定的酶或者准备好酶的保存液；
(3)准备好冻存的塑料冰袋或者冰盒备用；
(4)提前打开37℃水浴锅、低温离心机和制冰机。
1、瘤胃组织的机械分离
(1)在无菌操作台上，将无菌无RNA酶的细胞培养皿置于冰盒上，加入1×PBS；
(2)随即挑取三头体况和生产性能相近的健康奶牛，于瘤胃腹囊采集组织置于培养皿上，使用1×DPBS清洗2遍，记为样本一，样本二，样本三，为每个样本进行后续实验处理，作为平行实验(1)、平行实验(2)和平行实验(3)；图1、图2和图3分别是三个样本的单细胞悬液在显微镜下的图像：背景碎片较少，细胞结团情况几乎没有，细胞密度适中；
(3)观察肌层残留情况，使用灭菌手术剪刀镊子去除肌层，然后将组织块剪碎为10×0.5mm 2小块，转移至15ml EP管中，加入没过组织的20mM EDTA，于37℃浸泡30min；
(4)使用1×DPBS清洗2遍，将组织继续剪碎至1mm 3，弃上清，挑取适量组织块于新 的15ml EP管中；
2、牛瘤胃组织的酶解
(1)向含有1mm 2组织块的EP管中加入5ml 0.25％胰酶/EDTA，在37℃条件下消化5min；
(2)消化后迅速插入冰里2min，然后加入等体积预冷的HBSS，停止消化；在4℃、300g条件下离心2min，弃上清；
(3)加入2ml HBSS，300×g离心2min，再次清洗，弃上清；
(4)加入孵育好的2ml酶解离溶液(1.5mg/ml中性蛋白酶、1.5mg/ml胶原酶I、1.5mg/ml胶原酶IV、100U/ml透明质酸酶、50U/mlDNase I)，在37℃孵育30min；
(5)随后直接加入2ml 10％的FBS(V/V)，抑制酶活性；
3、杂质过滤
用30和70μm孔径的堆叠的细胞过滤器过滤消化好的细胞悬液，收集过滤好的细胞悬液于新的15ml EP管，在300×g、4℃条件下离心5min，弃上清。
4、细胞清洗
(1)向含有细胞沉淀的EP管中加入2ml HBSS，重悬细胞以清洗细胞，在300×g、4℃条件下离心5min，弃上清；
(2)再向EP管中加入2ml 1×PBS(含0.04％BSA)重悬细胞以清洗细胞，在300×g、4℃条件下离心5min，弃上清；
(3)然后再加入2ml 1×PBS(含0.04％BSA)重悬细胞。
5、再次过滤
(1)使用美天旎20μm细胞过滤器过滤消化好的细胞悬液，加入2ml 1×PBS(含0.04％BSA)冲洗滤膜，然后在300g、4℃条件下离心5min；
(2)观察细胞碎片情况，如果>10％，重复该步骤；
6、细胞观察和质检
(1)轻缓吹匀细胞悬液，取6μL左右置于血球计数板中，用显微镜观察背景碎片、细胞结团和细胞密度；如图4和图5所示，分别为平行实验(1)，(2)和(3)得到的牛瘤胃组织单细胞悬液的细胞浓度和细胞活率，样本一、二、三分别与图1、2、3中所使用样本相同：细胞浓度分别为1.15×10 6/mL，1.27×10 6/mL和2.64×10 6/mL，超过10 6/mL；细胞活率为分别为88％，94％和94％，达到85％的标准；
(2)加入台盼蓝染色，计算细胞活率、细胞浓度和细胞数目；
7、死细胞去除(选做)
(1)首先将专用的MS或者LS柱子、磁铁、架子、接收管放置在超净台上面准备去除 死细胞；
(2)在超净台内，使用双蒸水将20×Binding Buffer稀释成1×Binding Buffer(MS：3-4ml/rxn或者LS：17-26ml/rxn)；
(3)接着在300×g、4℃条件下离心，弃上清；
(4)收集细胞沉淀，加入100μL Dead Cell Removal MicroBeads吹打混匀，室温孵育15min；在孵育时间14min左右，加入1×Binding Buffer(MS:500μl；LS:3ml)；
(5)孵育结束之后，润洗液暴露柱子之前，向细胞悬液中加入1×Binding Buffer(MS:500-1000μl；LS:1-10ml)，并加入柱子上面；
(6)使用1×Binding Buffer(MS:4×500μl；LS:4×3ml)冲洗柱子；
(7)将收集管在300×g，4℃条件下离心，弃上清；
(8)然后加入合适体积的50-300μmL(根据细胞沉淀的多少判断加入的量)的0.04％BSA1×PBS重悬细胞；
(9)使用1ml移液枪吹打混匀5次，继续步骤(6)观察，直至细胞总量和细胞活性均达到要求。
8、质检
为进一步验证所获得牛瘤胃组织单细胞悬液的质量，进行cDNA以及library质检。如图6和图7所示，分别为牛瘤胃组织单细胞悬液的cDNA质检图和library质检图，cDNA质检图片段分布图的主峰值大于1000bp且无锯齿状杂峰，判定为及格；根据测序平台的要求，文库大小一般在300-600bp左右，其中包括了测序接头、read1、read2、10X barcode、UMI、Poly DT，插入片段以及样本index；在主峰450bp的情况下判定合格。
上述实施例用来解释说明本发明，而不是对本发明进行限制，在本发明的精神和权利要求的保护范围内，对本发明作出的任何修改和改变，都落入本发明的保护范围。

Claims (10)

  1. 一种用于单细胞测序的牛瘤胃上皮组织解离方法，其特征在于，具体包括以下步骤：
    (1)瘤胃组织的机械分离
    将无菌无RNA酶的细胞培养皿置于冰盒上，加入PBS；将瘤胃组织置于培养皿中，DPBS清洗后，使用灭菌手术剪刀镊子去除肌肉层，将组织块剪碎，放入EDTA浸泡，之后再次使用DPBS清洗，将组织继续剪碎，弃上清；
    (2)酶消化处理
    向步骤(1)最后剪碎的组织块中加入胰酶/EDTA消化；消化后插入冰里加入HBSS停止消化；离心后用HBSS再次清洗；加入孵育好的酶解离溶液，所述酶解离溶液包含中性蛋白酶、胶原酶I、胶原酶IV、透明质酸酶和DNase I，孵育结束后再离心；之后加入FBS抑制酶活性，得到消化好的细胞悬液；
    (3)杂质过滤
    用细胞过滤器过滤步骤(2)消化好的细胞悬液，收集至新的EP管；离心后弃上清；
    (4)细胞清洗
    向步骤(3)离心后的细胞沉淀中加入HBSS重悬，离心后弃上清；加入含有BSA的PBS进行重悬，离心后弃上清；再次加入含有BSA的PBS重悬细胞，得到细胞悬液；
    (5)再次过滤
    使用细胞过滤器过滤步骤(4)得到的细胞悬液，使用含有BSA的PBS冲洗滤膜后离心；观察细胞碎片情况，如果碎片率大于10％，重复该步骤；
    (6)细胞观察和质检
    吹匀步骤(5)得到的细胞悬液，取部分细胞悬液置于血球计数板中，用显微镜观察背景碎片、细胞结团和细胞密度；加入台盼蓝染色，计算细胞活性、细胞浓度和细胞总量。如果观察到细胞活性没有满足单细胞测序对细胞活性的要求，但细胞总量足量时需要进行死细胞去除操作。
  2. 如权利要求1所述的一种用于单细胞测序的牛瘤胃上皮组织解离方法，其特征在于，如果步骤(6)中观察到细胞活性没有满足单细胞测序对细胞活性的要求，但细胞总量足量时可使用去死细胞装置进行死细胞去除。具体操作如下：在超净台内，将20×Binding Buffer用双蒸水稀释成1×Binding Buffer；将步骤(5)得到的细胞悬液离心后弃上清；收集细胞沉淀，加入Dead Cell Removal MicroBeads吹打混匀；室温孵育，期间加入稀释后的Binding Buffer；孵育结束之后，润洗液暴露柱子之前，向细胞悬液中加入稀释后的Binding Buffer并 加入柱子上面；使用稀释后的Binding Buffer冲洗柱子；将收集管离心后弃上清；加入含BSA的PBS重悬细胞；吹打混匀，继续步骤(6)观察。
  3. 如权利要求1所述的一种用于单细胞测序的牛瘤胃上皮组织解离方法，其特征在于，步骤(1)中，首先将组织块剪碎成小块，体积为10×0.5mm 2，将组织继续剪碎时，剪碎至组织块体积为1mm 3。所述PBS为1×PBS，加入量为5ml；所述DPBS为1×DPBS。所述EDTA加入的体积与组织块体积之比为2：1。
  4. 如权利要求1所述的一种用于单细胞测序的牛瘤胃上皮组织解离方法，其特征在于，步骤(1)中，所述EDTA浓度为20mM，在37℃温度条件下浸泡30min。
  5. 如权利要求1所述的一种用于单细胞测序的牛瘤胃上皮组织解离方法，其特征在于，步骤(2)中，所述胰酶/EDTA的浓度为2500mg/L，加入的体积与将组织继续剪碎后的组织块之比为2：1；所述消化条件为：消化温度为37℃，消化时间为5min。冰里冷却时间为2min。所述HBSS体积为与细胞悬液相等体积，所述HBSS为预冷HBSS；所述离心条件为：离心转速为300×g，离心温度为4℃，离心时间为2min。所述酶解离溶液加入的体积与将组织继续剪碎后的组织块之比为2：1，其包含1.5mg/ml中性蛋白酶、1.5mg/ml胶原酶I、1.5mg/ml胶原酶IV、100U/ml透明质酸酶、50U/mlDNase I；反应条件为：孵育温度为37℃，孵育时间为30min。所述FBS为10％FBS(V/V)，体积与加入酶解离溶液体积相同。
  6. 如权利要求1所述的一种用于单细胞测序的牛瘤胃上皮组织解离方法，其特征在于，步骤(3)中，所述过滤使用30μm和70μm孔径的堆叠的细胞过滤器。所述离心条件为：离心转速为300×g，离心温度为4℃，离心时间为5min。
  7. 如权利要求1所述的一种用于单细胞测序的牛瘤胃上皮组织解离方法，其特征在于，步骤(4)中，所述HBSS的加入量为2mL。所述PBS的加入量为2mL；所述PBS为1×PBS，所述PBS含有浓度为400mg/L的BSA。所述离心条件均为：离心的转速为300g，离心的温度为4℃，离心的时间为5min。
  8. 如权利要求1所述的一种用于单细胞测序的牛瘤胃上皮组织解离方法，其特征在于，所述步骤(5)中，所述细胞过滤器使用美天旎20μm细胞过滤器；所述PBS为1×PBS，所述PBS含有浓度为400mg/L的BSA。所述离心条件均为：离心的转速为300g，离心的温度为4℃，离心的时间为5min。
  9. 如权利要求2所述的一种用于单细胞测序的牛瘤胃上皮组织解离方法，其特征在于，所述去死细胞装置包括MS或LS柱子、磁铁、架子、接收管。对于MS或LS，Binding Buffer使用量不同，所述稀释后的Binding Buffer为1×Binding Buffer；MS的使用条件为3-4ml/rxn，LS的使用条件为17-26ml/rxn。孵育时Binding Buffer加入量为：MS中加入500μl，LS中加 入3ml；孵育结束后，Binding Buffer的加入量为：MS中加入500-1000μl；LS中加入1-10ml。冲洗柱子时，Binding Buffer的加入量为：MS中每次500μl，共4次；LS中每次3ml，共4次。
  10. 如权利要求2所述的一种用于单细胞测序的牛瘤胃上皮组织解离方法，其特征在于，所述Dead Cell Removal MicroBeads的添加量为100μl。所述孵育的时长为15min，加入Binding Buffer的时间为孵育的第14min；所述离心条件均为：离心的转速为300×g，离心的温度为4℃，离心的时间为5-10min。所述PBS的加入量为50-300μl；所述PBS为1×PBS，所述PBS含有浓度为400mg/L的BSA。
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