CN113293196A - Single cell nucleus extraction method suitable for frozen tissue - Google Patents

Single cell nucleus extraction method suitable for frozen tissue Download PDF

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CN113293196A
CN113293196A CN202110855298.4A CN202110855298A CN113293196A CN 113293196 A CN113293196 A CN 113293196A CN 202110855298 A CN202110855298 A CN 202110855298A CN 113293196 A CN113293196 A CN 113293196A
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cell nucleus
nuclear
nebcs
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訾晓渊
尤园园
朱文奇
丁修恒
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Nanjing Tongyuan Medical Laboratory Co ltd
Singleron Nanjing Biotechnologies Ltd
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    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1017—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by filtration, e.g. using filters, frits, membranes

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Abstract

The invention relates to the technical field of biology, in particular to a single cell nucleus extraction method suitable for frozen tissues. The invention uses the NEBCS nuclear separating fluid to successfully separate and extract the mononuclear cell nucleus from the frozen tissue in a short time, and obtains the purer mononuclear cell nucleus through differential centrifugation. The proportion of degradation of nuclear RNA is greatly reduced, so that a higher proportion of protein coding RNA can be obtained at the single cell level, and the mitochondrial gene proportion is effectively reduced. The single cell nucleus extraction method provided by the invention is suitable for various frozen tissues, and can effectively prevent further degradation of the RNA method. The operation process is simple, the extraction process of the cell nucleus can be completed by using common laboratory instruments such as a centrifuge, a microscope and the like, and the whole process only needs 30-60 min. Special instruments and equipment are not needed, the extraction cost is effectively reduced, and the practicability is high.

Description

Single cell nucleus extraction method suitable for frozen tissue
Technical Field
The invention relates to the technical field of biology, in particular to a single cell nucleus extraction method suitable for frozen tissues.
Background
The single cell complete transcriptome sequencing technology is one new single cell level complete transcriptome amplifying and sequencing technology. The principle is that the separated micro total transcriptome RNA of a single cell is amplified and then subjected to high-throughput sequencing, and the method is a tool for researching the cell type, the dynamic state and the functional process in a complex tissue. Compared with the traditional transcriptome sequencing, the single cell sequencing not only can measure the gene expression level more accurately, but also can detect trace gene expression, and has the advantages of omnibearing and multilevel. However, many precious human tissue samples are stored in frozen tissue form, and living cells obtained after digestion by enzymatic methods are very few and cannot be used for sequencing single-cell transcriptome. At present, the difficult problem of frozen tissue sequencing can be solved and a plurality of obvious advantages are achieved through mechanical extraction of cell nuclei, but the process of extracting the cell nuclei by the existing mechanical extraction method is complicated, the requirements on reagent instruments and the like are high, if iodixanol is needed or the cell nuclei are separated by a flow cytometer, experiments can not be developed in a common laboratory, and the cell nucleus extraction cost is greatly increased.
Disclosure of Invention
In view of the above, the present invention provides a method for extracting single cell nuclei suitable for frozen tissues, which overcomes the disadvantages of the prior art. The single cell nucleus extraction method provided by the invention reduces the degradation of RNA to the maximum extent, and solves the problems of long operation time, complicated flow, high cost and the like in the existing single cell nucleus extraction from frozen tissues.
In order to solve the technical problems, the technical scheme provided by the invention is as follows:
the invention provides a method for extracting single cell nucleus of frozen tissue, which comprises the following steps:
(1) taking the frozen tissue into a sterile centrifuge tube, placing the sterile centrifuge tube in an ice bath, adding a small amount of precooled NEBCS nuclear separating medium, immediately shearing the frozen tissue into pieces, adding the NEBCS nuclear separating medium again, and incubating on ice;
(2) after the incubation is finished, filtering the mixture into a centrifuge tube by using a filter, and washing the filter by using PBS-RI buffer solution; centrifuging the supernatant again after the first centrifugation, and fully removing the supernatant; adding PBS-RI buffer solution to resuspend the cell nucleus sediment, adding DAPI cell nucleus dye solution, mixing uniformly, and standing in the dark; and (3) washing cell nuclei by using PBS-RI buffer solution, centrifuging to remove supernatant, re-suspending the cell nuclei by using the PBS-RI buffer solution, and diluting to obtain cell nucleus suspension.
Further, the composition of the NEBCS nuclear separation solution in the step (1) comprises 0.32M sucrose and 5 mM CaCl2，3 mM Mg(Ac)2，0.1 mM EDTA，20 mM Tris-HCl，0.05%~0.08% Triton X-100，5 mM ~8mM DTT , 0.2 U/μL RNase inhibitor。
The incubation time in the step (1) is 5-10 min.
In the step (2), the diameter of the filter is 40-50 um.
The PBS-RI buffer solution in the step (2) contains 5 mM DTT and 0.02U/. mu.L RNase inhibitor.
Preferably, the PBS-RI buffer is pre-cooled in a 4 ℃ environment before use.
In the step (2), the rotating speed of the first centrifugation is 100-300 rcf, and the centrifugation time is 1-3 min; the speed of re-centrifugation was 500 rcf and the time of centrifugation was 5 min.
The concentration of the cell nucleus suspension in the step (2) is 3-5 multiplied by 105 nucleus/mL。
The invention also provides an NEBCS nuclear separation liquid, which is an optimized NEB nuclear separation liquid and comprises the components of 0.32M sucrose and 5 mM CaCl2，3 mM Mg(Ac)2，0.1 mM EDTA，20 mM Tris-HCl，0.05%~0.08%Triton X-100，5 mM~8mM DTT，0.2 U/μL RNase inhibitor。
Preferably, the composition of the NEBCS nuclear separation liquid comprises 0.32M sucrose and 5 mM CaCl2，3 mM Mg(Ac)2，0.1 mM EDTA，20 mM Tris-HCl，0.05%Triton X-100，5 mM DTT，0.2U/μL RNase inhibitor。
The NEBCS separating medium provided by the invention reduces the use concentration of Triton-X100, and the DTT and the RNase inhibitor are added, so that the integrity of cell nucleus can be protected while cell membranes are effectively cracked, and the degradation of RNA can be effectively inhibited.
The invention also provides the application of extracting the cell nucleus according to the single cell nucleus extraction method of the frozen tissue and performing single cell omics analysis on the cell nucleus.
Compared with the prior art, the invention has the beneficial effects that:
the invention uses the NEBCS nuclear separating fluid to successfully separate and extract the mononuclear cell nucleus from the frozen tissue in a short time, and then obtains the purer mononuclear cell nucleus through differential centrifugation. The proportion of degradation of the nuclear RNA is greatly reduced, the single cell level can obtain protein coding RNA with higher proportion, the ratio of mitochondrial genes is effectively reduced, and the self-control of cells is effectively eliminated. The single cell nucleus extraction method provided by the invention is suitable for various frozen tissues, and can effectively prevent further degradation of the RNA method. The operation process is simple, the extraction process of the cell nucleus can be completed by using common laboratory instruments such as a centrifuge, a microscope and the like, and the whole process only needs 30-60 min. Special instruments and equipment are not needed, the extraction cost is effectively reduced, and the practicability is high.
Detailed Description
The invention discloses a method for extracting single cell nuclei suitable for frozen tissues. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. The experimental procedures, in which specific conditions are not specified in the examples below, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.
Sample source: human frozen bone cancer tissues and mouse frozen liver tissues are both from tumor hospitals in Jiangsu province.
Example 1
This example configures 10 mL of a NEBCS nuclear separation medium, and separately pipettes 1 mL of 3.2M sucrose, 100. mu.L of 500 mM CaCl2，100 μL 300 mM Mg(Ac)2100 mu L of 10mM EDTA, 100 mu L of 2M Tris-HCl, 80 mu L of 10% Triton X-100, 800 mu L of 100 mM DTT and 50 mu L of 40U/. mu.L RNase inhibitor were added in a 15 mL EP tube, and the volume was made up to 10 mL using nuclease-free water to obtain the NEBCS nuclear separation medium.
Example 2
This example configures 10 mL of a NEBCS nuclear separation medium, and separately pipettes 1 mL of 3.2M sucrose, 100. mu.L of 500 mM CaCl2，100 μL 300 mM Mg(Ac)2100 mu L of 10mM EDTA, 100 mu L of 2M Tris-HCl, 50 mu L of 10% Triton X-100, 500 mu L of 100 mM DTT and 50 mu L of 40U/. mu.L RNase inhibitor were added to a 15 mL EP tube, and the volume of the tube was made up to 10 mL using nuclease-free water to obtain the NEBCS nuclear separation medium.
Example 3
In this example, the NEBCS nuclear separation fluid prepared in example 1 was used to extract individual nuclei of human cryopreserved osteocarcinoma tissues, and the specific extraction steps were as follows:
(1) putting 50mg of human cryopreserved bone cancer tissues into a 1.5 mL sterile centrifuge tube, placing the tube in an ice bath, adding 100 mu L of 4 ℃ precooled NEBCS nuclear separation liquid, immediately shearing the bone cancer tissues into minced substances by using surgical scissors, adding 1 mL of NEBCS nuclear separation liquid, placing the minced substances on ice for incubation for 5-10 min, and reversing and uniformly mixing the mixture every 2 min for 1 time;
(2) after the incubation is finished, filtering the mixture into a 15 mL centrifuge tube by using a 50um filter, and washing the filter by using 4 mL precooled PBS-RI buffer solution; centrifuging at 100-300 rcf at 4 ℃ for 1-3 min, sucking 4.5 mL of supernatant, and transferring to a new 15 mL centrifuge tube; centrifuging at 500 rcf for 5 min, sufficiently removing supernatant, adding 50 μ L of precooled PBS-RI buffer solution to resuspend cell nucleus precipitate, adding 200 μ L of DAPI cell nucleus dye solution, mixing uniformly, and standing in dark for 2 min; adding 2 mL of precooled PBS-RI buffer solution again to wash the cell nucleus, centrifuging for 5 min at 500 rcf, fully removing the supernatant, and adding 200 mu L of precooled PBS-RI buffer solution to resuspend the cell nucleus;
(3) taking 10 mu L of cell nucleus suspension, counting under a microscope by using a blood cell counter, calculating the cell nucleus concentration, and adjusting the cell nucleus concentration to 3-5 multiplied by 105core/mL.
Example 4
In this example, the NEBCS nuclear separation medium configured in example 2 was used to extract single nuclei from mouse liver tissue, and the single-nucleus suspension was used in the GEXSCOPE microfluidic single-cell analysis platform for single-cell nuclear transcriptome analysis. The specific extraction steps are as follows:
(1) freezing and storing the liver tissue of a 100 mg rat into a 1.5 mL sterile centrifuge tube, placing the tube in an ice bath, adding 100 mu L of 4 ℃ precooled NEBCS nuclear separation liquid, shearing the liver tissue of the rat into a minced substance, adding 1 mL of NEBCS nuclear separation liquid, placing the minced substance on ice for incubation for 5 min to 10min, and reversing and mixing the mixture for 1 time every 2 min;
(2) after the incubation is finished, filtering the mixture into a 15 mL centrifuge tube by using a 40 um filter, and washing the filter by using 4 mL precooled PBS-RI buffer solution; centrifuging at 200-300 rcf at 4 ℃ for 1-5 min, temporarily retaining the precipitate, sucking the supernatant, centrifuging at 500 rcf for 5 min, sufficiently removing the supernatant, adding 50 mu L of precooled PBS-RI buffer solution to resuspend the cell nucleus precipitate, adding 200 mu L of DAPI cell nucleus dye solution, uniformly mixing, and standing in the dark for 2 min; adding 2 mL of precooled PBS-RI buffer solution again to wash the cell nucleus, centrifuging for 5 min at 500 rcf, fully removing the supernatant, and adding 200 mu L of precooled PBS-RI buffer solution to resuspend the cell nucleus;
(3) taking 10 μ L of cell nucleus suspension, counting under microscope with hematocytometer, calculating cell nucleus concentration, and adjusting cell nucleus concentration to 4 × 105core/mL. For subsequent analysis of single-cell nuclear transcriptome.
Example 5
Nuclei in 100 mg of mouse cryopreserved liver tissue were extracted in the same manner as in example 4, except that the same volume of NEB nuclear fraction was used instead of NEBCS nuclear fraction in step (1). The NEB nuclear separation liquid comprises the following components: 0.32M sucrose, 5 mM CaCl2，3 mM Mg(Ac)2，0.1 mM EDTA，20 mM Tris-HCl，0.1 %Triton X-100。
Example 6
Nuclei from 100 mg of rat cryopreserved liver tissue were extracted in the same manner as in example 4 except that the NEBCS nuclear isolate was replaced with NST/DAPI buffer in step (1). The NST/DAPI buffer solution comprises the following components: NST/DAPI buffer 800 mL NST (146 mM NaCl, 10mM Tris base pH7.8, 1 mM CaCl2, 21 mM MgCl20.05% BSA, 0.2% Nonidet P-40), 200 mL of 106 mM MgCl210 mg of DAPI and 5 mM of EDTA.
Example 7
In this example, in order to compare the extraction effect of single cell nuclei, the single cell nuclei suspension obtained in examples 4 to 6 was respectively used for single cell nucleus transcription group analysis by using a GEXSCOPE microfluidic single cell analysis platform. The experiments were divided into three groups, NEBCS (example 4), NEB (example 5) and NST (example 6).
Before the single cell nuclear transcription experiment was started, the test bed was sprayed once with RNase away and wiped clean. And (3) absorbing the redundant liquid at the big mouth end, adding 200 mu L PBS to rinse the chip, and absorbing the redundant liquid at the big mouth end. Gently mixing the cell nucleus suspension, sucking 100 μ L of cell nucleus suspension, adding into the sample inlet, controlling the loading process at 40 s, and sucking off the excess liquid at both ends. And (4) flushing redundant cell nucleuses by 400 mu L of PBS, observing whether the redundant cell nucleuses adhered to the surface are removed under a microscope, if the redundant cell nucleuses are remained, continuously flushing by PBS, and sucking redundant liquid at two ends after flushing in each step. Add 50. mu.L of resuspended beads, gently pipette the beads, and pump the beads from the sample inlet. PBS was added to the chip until the beads were spread over the entire chip. Excess beads were washed with PBS. The number of wells in which no beads fell in 3 fields of view was counted (or photographed), and data was recorded.
100 μ L of lysate was pipetted slowly into the sample inlet (30 sec) and allowed to stand at room temperature for 20 min to lyse the cells and release the mRNA, allowing the beads to capture the mRNA. Add 8 mL of 6XSSC and 8. mu.L of 20% Tween solution to a 15 mL centrifuge tube, vortex and mix well, and place on ice. A sterile petri dish and a pair of forceps were prepared for reverse transcription by RNase away. And (3) spraying RNase away with two hands, detaching the chip, and putting PDMS in the chip into a corresponding 15 mL centrifuge tube. Vortex the tube 2 times for 5 min each to allow beads to fall out of the wells. Centrifuge at 1000 rcf for 1 min, aspirate bottom beads and transfer to a 1.5 mL centrifuge tube. Centrifuge, 1000 rcf, 1 min, discard supernatant. Add 200. mu.L of 1 xwash buffer to wash 1 time, centrifuge, 4 ℃, 1000 rcf, 1 min, discard the supernatant. Instantaneously, suck the liquid as clean as possible, leaving only the bottom beads. RT mix (multi-mix 10%) was formulated on ice, vortexed and centrifuged briefly. The proportioning components of RT mix are shown in Table 1:
TABLE 1 RT mix ratio
RT mix Vol
2× master mix 100 μL
DTT 20 μL
TS1 5 μL
Rnase inhibitor 5 μL
P7600 10 μL
NFW 60 μL
200 μ L of prepared RT mix was added into a centrifuge tube containing beads, and the mixture was placed in a pre-set oven and mixer at 42 ℃ for 90 min. After completion of the RT reaction, the tube was centrifuged at 1000 rcf for 1 min and the supernatant was discarded. The beads were washed with 1 mL of 0.5% TE-SDS, centrifuged, 1000 rcf, 1 min, and the supernatant was discarded. The beads were washed with 1 mL of 0.01% TE-TW, centrifuged, 1000 rcf, 1 min, and the supernatant discarded. Washed 2 times with 1 mL NF water, centrifuged, 1000 rcf, 1 min, and the supernatant discarded. Instantaneously, the liquid is sucked up as clean as possible, and only the bottom beads are left for the next PCR. Each chip was prepared as a PCR mix according to 6.1 reactions, formulated as a PCR system, vortexed, mixed and flash isolated. The formulation of the PCR mix is shown in Table 2:
TABLE 2 PCR mix
PCR mix Vol
5× HiFi fidelity buffer 10μL
dNTP mix 1.5 μL
Hot STAR 1 μL
SP1 0.4 μL
NFW 37.1 μL
And adding the PCR mix into the centrifuge tube with the beads in the previous step, blowing 305 mu L of each tube, and uniformly mixing. Adding the mixture into an 8-row calandria, covering a pipe cover by a system with 50 mu L of each hole, and setting a PCR program as follows: lid 105 ℃, 1) 95 ℃ for 3min, 2) 98 ℃ for 20 sec, 3) 65 ℃ for 45 sec, 4) 72 ℃ for 3min, 4 cycles, 5) 98 ℃ for 20 sec, 6) 67 ℃ for 20 sec, 7) 72 ℃ for 3min, 12 cycles, 8) 72 ℃ for 5 min, 9) 12 ℃ hold. And placing the mixture in a PCR instrument for amplification.
The product purification is carried out by using VAHTS RNA Clean Beads, and the specific operation method is shown in the specification. Fragment analysis was performed using an AATI DNA fragment analyzer. The library was created using the novifading library kit (TD 502) and 2 generations of sequencing were performed based on the illumina platform. Sequencing results were analyzed using a single cell analysis procedure developed autonomously for new format, and table 3 is a summary table of sequencing data analysis.
TABLE 3 sequencing data analysis summary sheet
Sample name NEBCS NEB NST
Rawreads 2733191 4305903 3995105
Valid_Barcodes 84.60% 83.31% 87.45%
Cell_BarcodeQ30 98.39% 98.13% 98.05%
UMIQ30 98.18% 98.12% 97.93%
Filter_reads 420886(15.39%) 718273(16.68%) 501371(12.54%)
Low_quality 49882(2.15%) 182552(5.08%) 234585(6.71%)
Too_many_N 1210(0.05%) 339(0%) 315(0%)
Too_short 70301(3.04%) 122867(3.42%) 60349(1.72%)
Adapter 823469 1613180 1165771
rawreads 2312305 3587630 3493734
database 0.346 0.538 0.524
rawQ20 88.83 82.95 85.13
rawQ30 81.51 73 75.13
cleanreads 2190912 3281872 3198485
cleandata 0.262 0.362 0.389
Q20 95.8 93.79 93.68
Q30 91 86.88 86.08
clean_rate 75.72 67.28 74.23
Uniquely_mapped 1757650(80.22%) 2609353(79.51%) 2670918(83.51%)
MμLtiple_mapped 172409(7.87%) 281676(8.58%) 277747(8.68%)
exon 35.49% 52.20% 61.88%
intron 56.29% 34.73% 29.21%
intergenic 8.22% 13.08% 8.91%
Assigned 1481281(84.27%) 1981547(75.94%) 2213500(82.87%)
Unassigned_NoFeatures 228096(12.97%) 563029(21.57%) 393262(14.72%)
Unassigned_Ambiguity 48273(2.74%) 64777(2.48%) 64156(2.4%)
Cells_number 2133 2976 3499
Cells_reads_PCT 50.82% 46.58% 29.51%
Saturation 25.95% 26.17% 17.50%
Mean_Reads.Cell 1281 1446 1141
Median_UMIs 214 175 130
Total_Genes 16719 22376 20714
Median_Genes 178 143 92
mito_percent (>20%) 4.594468 9.509409 63.64676
As can be seen from Table 3, the single cell nucleus extraction method provided by the invention can avoid the degradation of RNA, so that a higher proportion of protein coding RNA can be obtained at the single cell level, and the mitochondrial gene ratio is effectively reduced. Has strong practicability.
It should be understood that the series of detailed descriptions set forth above are merely specific descriptions of possible embodiments of the present invention and are not intended to limit the scope of the present invention, and equivalent embodiments or modifications made without departing from the technical spirit of the present invention should be included in the scope of the present invention.

Claims (8)

1. A method for extracting single cell nuclei from frozen tissues is characterized by comprising the following steps:
(1) taking the frozen tissue into a sterile centrifuge tube, placing the sterile centrifuge tube in an ice bath, adding a small amount of precooled NEBCS nuclear separating medium, immediately shearing the frozen tissue into pieces, adding the NEBCS nuclear separating medium again, and incubating on ice;
(2) after the incubation, filtration was carried outFiltering the mixture into a centrifuge tube by using a device, and washing the filter by using PBS-RI buffer solution; centrifuging the supernatant again after the first centrifugation, and fully removing the supernatant; adding PBS-RI buffer solution to resuspend the cell nucleus sediment, adding DAPI cell nucleus dye solution, mixing uniformly, and standing in the dark; washing cell nuclei by PBS-RI buffer solution, centrifuging to remove supernatant, re-suspending the cell nuclei by the PBS-RI buffer solution, and diluting to obtain cell nucleus suspension; the composition of the NEBCS nuclear separation liquid in the step (1) comprises 0.32M sucrose and 5 mM CaCl2，3 mM Mg(Ac)2，0.1 mM EDTA，20 mM Tris-HCl，0.05%~0.08%Triton X-100，5 mM ~8mM DTT，0.2 U/μL RNase inhibitor。
2. The extraction method according to claim 1, wherein the incubation time in step (1) is 5-10 min.
3. The extraction method according to claim 1, wherein the diameter of the filter in the step (2) is 40-50 um.
4. The extraction method according to claim 1, wherein the PBS-RI buffer solution in step (2) contains 5 mM DTT and 0.02U/. mu.L RNase inhibitor at the final concentration.
5. The extraction method according to claim 1, wherein the rotation speed of the first centrifugation in the step (2) is 100-300 rcf, and the centrifugation time is 1-3 min; the speed of re-centrifugation was 500 rcf and the time of centrifugation was 5 min.
6. The extraction method according to claim 1, wherein the concentration of the cell nucleus suspension in the step (2) is 3 to 5 x 105 nucleus/mL。
7. The NEBCS nuclear separation liquid is characterized in that the NEBCS nuclear separation liquid is optimized NEB nuclear separation liquid, and the composition of the NEBCS nuclear separation liquid comprises 0.32M sucrose and 5 mM CaCl2，3 mM Mg(Ac)2，0.1 mM EDTA，20 mM Tris-HCl，0.05%~0.08%Triton X-100，5 mM~8mM DTT，0.2 U/μL RNase inhibitor。
8. A single cell nuclear sequencing method, characterized in that the extraction method according to any one of claims 1 to 6 is used for extracting cell nuclei and performing single cell omics analysis on the cell nuclei.
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CN114015752A (en) * 2021-10-26 2022-02-08 上海欧易生物医学科技有限公司 Mouse retina frozen tissue cell nucleus suspension suitable for single cell sequencing and preparation method and application thereof
CN114015752B (en) * 2021-10-26 2023-08-22 上海欧易生物医学科技有限公司 Mouse retina frozen tissue cell nucleus suspension suitable for single cell sequencing and preparation method and application thereof
CN114350610A (en) * 2021-12-21 2022-04-15 北京百迈客生物科技有限公司 Kit for separating brain and spinal cord tissue cell nucleus and cell nucleus separation method
CN114350610B (en) * 2021-12-21 2023-11-28 北京百迈客生物科技有限公司 Kit for separating brain and spinal cord tissue cell nuclei and cell nucleus separation method
CN117106698A (en) * 2023-10-24 2023-11-24 中国海洋大学三亚海洋研究院 Method for extracting liver and gill tissue cell nuclei of marine fish
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