CN109628555A - People suitable for unicellular sequencing freezes tumor tissue cell's core separation method - Google Patents
People suitable for unicellular sequencing freezes tumor tissue cell's core separation method Download PDFInfo
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Abstract
The invention discloses the methods that the people for being suitable for unicellular sequencing freezes tumor tissue cell's core separation method and extracts nucleus.The method of the extraction nucleus includes: that sample to be extracted is carried out cracking processing, and the cracking processing is carried out in the first buffer；Pyrolysis product is subjected to the first centrifugal treating, to obtain nucleus precipitating；Nucleus precipitating is rinsed and resuspension is handled, rinses and resuspension processing is carried out in the second buffer, to obtain the nucleus；Wherein, first buffer includes: 8mM Tris alkali, the 0.8mM CaCl of 116.8mM NaCl, 7.8 pH2、38mM MgCl2, 0.04%BSA, 0.16%Nonidet P-40,1mM EDTA, 1mg/mL DAPI；Second buffer includes: 1 × PBS, 1.0%BSA and 0.2U/ μ L RNase inhibitor.This method only needs simple reagent consumptive material, does not need expensive separation equipment, and enough, complete monokaryon can be obtained from less frost tumor tissues and is sequenced for unicellular RNA.
Description
Technical field
The present invention relates to sequencing fields, in particular it relates to which the people for being suitable for unicellular sequencing freezes tumor tissues
Nucleus separation method.
Background technique
Unicellular sequencing refers in individual cells level, carries out high-flux sequence to genome, transcript profile, apparent group etc.
The new technology of analysis reflects intercellular heterogeneity for disclosing the gene structure and gene expression status of individual cells, 2013
Annual Technical is chosen as year by Nature Methods, Science is classified as the six big field umber ones most to merit attention in year, be 2017
The key technology of year human cell's map intended application.Unicellular sequencing can be applied to Developmental Biology, immunology, nervous system
The research in the directions such as cell, tumor tissues, wherein it is relevant to carry out Tumor Heterogeneity using extensive unicellular transcript profile sequencing
Research has become popular direction.Unicellular transcript profile sequencing is sequenced by the RNA to the individual cells in tumor tissues, point
The expression conditions in each cell are analysed, classification comparison is carried out to cell, studies tumor development mechanism, drug effect machine
System etc. provides new direction for tumor classification, ideas of cancer therapy, drug efficacy prediction, new drug development etc..
It is differentiated to carry out separate marking to the RNA in each cell for data, the cell before label is completed must be
Single suspension and complete living cells, therefore unicellular sequencing is more demanding to sample, including cell suspended state, concentration, work
Rate etc..Human tumour tissue is not easy to obtain high motility rate single cell suspension, and single cell suspension is not easy to transport, because of nothing in transportational process
Method guarantees Cell viability.Current implementation method is to carry unicellular separation equipment to hospital, and the flesh tissue cut of performing the operation is vertical
Carry out digestion and subsequent separating treatment, make the experiment cost, the time, in terms of by larger limitation.Therefore have
Necessity develops a kind of new sample processing method, more easily obtains suitable sample and carries out unicellular transcript profile sequencing.
It can be transported and be saved with dry ice after being frozen tissue with liquid nitrogen frozen for flesh tissue, but be difficult to digest after thawing and obtain
Obtain the single cell suspension of high motility rate.Cell membrane can be caused during flesh tissue fast freezing and freezen protective biggish
Damage, the free loss of intracellular rna, leads to not carry out unicellular RNA sequencing, and nuclear membrane can be kept during freeze thawing
Completely.There are some researches prove the RNA in nucleus including sufficient amount, can be used for being sequenced, and have with RNA sequencing is carried out with intact cell
There is higher correlation.Since nuclear membrane is easier to keep complete than cell membrane, compared with obtaining single cell suspension, from frozen tissue
The operability for obtaining nucleus suspension is stronger.Obtaining monokaryon suspension from frozen tissue at present, there are no mature and stable sides
Method at present only by expensive ancillary equipment for the monokaryon suspension separation method of frost brain tissue, and does not have Quality Control result
Show.With the extensive development of immunotherapy of tumors, unicellular sequencing can carry out tumor microenvironment going deep into system
System is researched and analysed including predicting effective crowd to immunization therapy.
Thus, a kind of simply and easily separation people frost tumor tissue cell's core is developed, the slender karyon of high motility rate is obtained
The method of suspension is extremely urgent.
Summary of the invention
The application is made based on more than inventor true and problem discovery and understanding:
The present invention is directed to solve at least some of the technical problems in related technologies.For this purpose, inventor opens
A kind of method for having sent out simplicity can obtain the monokaryon suspension suitable for unicellular sequencing, solution from frost tumor tissues sample
Limitation except the unicellular sequencing of fresh tumor tissue to time and location, is applied more broadly in unicellular sequencing technologies swollen
The research of tumor microenvironment.
In the first aspect of the present invention, the invention proposes a kind of methods for extracting nucleus.Implementation according to the present invention
Example, which comprises sample to be extracted is subjected to cracking processing, the cracking processing is carried out in the first buffer；
Pyrolysis product is subjected to the first centrifugal treating, to obtain nucleus precipitating；Nucleus precipitating is rinsed and is resuspended
Processing, is rinsed and resuspension processing is carried out in the second buffer, to obtain the nucleus；Wherein, described first is slow
Fliud flushing includes: 8mM Tris alkali, the 0.8mM CaCl of 116.8mM NaCl, 7.8 pH2、38mM MgCl2, 0.04%BSA (%
It is mass volume ratio, unit g/100mL, such as the configurable 0.04%BSA of addition 0.04g BSA in 100mL buffer),
(% is volume ratio to 0.16%Nonidet P-40, unit mL/100mL, such as 0.16mL is added in 100mL buffer
Nonidet P-40 can configure 0.04%Nonidet P-40), 1mM EDTA, 1mg/mL DAPI；The second buffer packet
Include: (% is mass volume ratio, unit g/100mL, such as 1g is added in 100mL buffer by 1 × PBS, 1.0%BSA
BSA can configure 1.0%BSA) and 0.2U/ μ L RNase inhibitor.Wherein, in the present invention, the first buffer is also known as NST
Lysis buffer, is prepared as follows, and merges mixing: 1) 80mL of NST after preparing following two components respectively:
146mM NaCl,10mM Tris base at pH 7.8,1mM CaCl2,21mM MgCl2, 0.05%BSA, 0.2%
Nonidet P-40)；2) the 106mM MgCl of 20mL2, 5mM EDTA, 1mg/mL DAPI.Inventors have found that the second buffer
In BSA presence, the integrality of nuclear membrane can be protected.Simple reagent consumptive material is only needed according to the method for the embodiment of the present invention, no
Need expensive separation equipment, so that it may enough, complete monokaryon is obtained from less frost tumor tissues for unicellular
RNA sequencing, and then nucleus suspension obtained is sequenced suitable for unicellular RNA according to the method for the embodiment of the present invention, can answer
For being difficult to obtain the research such as flesh tissue, Tumor Heterogeneity that unicellular level can be carried out with the sample that frozen tissue replaces.
According to an embodiment of the invention, the above method can further include at least one following additional technical feature:
According to an embodiment of the invention, first buffer and the second buffer first pass through 4 DEG C of precooling treatments in advance.
According to an embodiment of the invention, the sample to be extracted be frost tumor tissues, the sample to be extracted with it is described
The mass volume ratio of first buffer is 30mg:3mL.In turn, sample dissociation can be made more abundant.
According to an embodiment of the invention, the cracking processing carries out in the following way: by the sample to be extracted
Mechanical Crushing processing is carried out in first buffer, the sample size after break process is not more than 1mm3；By Mechanical Crushing
Processing product ice sets processing 30min, and every 5min is mixed；Ice is set into processing product and carries out 10~15 piping and druming processing, and will be described
Piping and druming processing product carries out the first filtration treatment, and first filtration treatment is that the cell filter for being 37 μm by diameter carries out
, the filtrate after first filtration treatment constitutes the pyrolysis product.Inventors have found that the sample size after break process is big
In 1mm3, then will lead to cracking it is insufficient, the core rate of recovery is low.
According to an embodiment of the invention, first centrifugal treating is to carry out 5min under conditions of 4 DEG C, 500g.As a result,
The rate of recovery of pyrolysis product can be increased under conditions of not influencing nuclear membrane integrality.
According to an embodiment of the invention, the flushing and resuspension processing carry out in the following way: by the cell
Core, which is deposited in second buffer, carries out the first resuspension processing, and the dosage of second buffer is 5mL；First is resuspended
It handles product and carries out the second filtration treatment；The filtrate of second filtration treatment is subjected to the second centrifugal treating, at second centrifugation
Reason is to carry out 5min under conditions of 4 DEG C, 500g；Precipitating after second centrifugal treating is carried out second, processing and third is resuspended
Filtration treatment.
According to an embodiment of the invention, re-suspension liquid is carried out pressure-vaccum 10 times repeatedly in first resuspension processing.Thus
Nucleus precipitating can be uniformly mixed with the second buffer.
According to an embodiment of the invention, second, third described filtration treatment is the cell filter for being 37 μm by diameter
It carries out.Cell fragment and tissue mass can be filtered out as a result,.
According to an embodiment of the invention, further comprising will be after third filtration treatment after the flushing and resuspension processing
Filtrate carries out gradient centrifugation processing.
According to an embodiment of the invention, the gradient centrifugation is carried out in Sucrose buffer I.
According to an embodiment of the invention, the Sucrose buffer I includes: 1.35ml Nuclei PURE 2M sucrose cushion
Liquid and 150 μ l Nuclei PURE buffers.
According to an embodiment of the invention, the gradient centrifugation carries out in the following way: at toward the third filtering
The Sucrose buffer I is added in filtrate after reason, pressure-vaccum mixes 10 times；Filtrate after pressure-vaccum is mixed 10 times is added to described
The upper layer Sucrose buffer I, not mix, to obtain layering liquid；By the layering liquid temperature be 4 DEG C, speed 13000g
Under conditions of centrifugal treating 45min；Product after centrifugal treating is removed into supernatant, to obtain the nucleus.It as a result, can be into one
The impurity such as step removal cell fragment.
In the second aspect of the present invention, the invention proposes a kind of methods for extracting frost tumor tissue cell's core.According to
The embodiment of the present invention, comprising: frost tumor tissues are subjected to Mechanical Crushing processing in the first buffer, after break process
Sample size is not more than 1mm3；First buffer first passes through 4 DEG C of precooling treatments in advance, the frost tumor tissues and described the
The mass volume ratio of one buffer is 30mg:3mL；Mechanical Crushing processing product ice is set into processing 30min, every 5min is mixed；It will
Ice sets processing product and carries out 10~15 piping and druming processing, and by piping and druming processing product the first filtration treatment of progress, and described the
One filtration treatment is that the cell filter for being 37 μm by diameter carries out, and the filtrate after first filtration treatment constitutes cracking
Product；Pyrolysis product is subjected to the first centrifugal treating, first centrifugal treating is to carry out 5min under conditions of 4 DEG C, 500g,
To obtain nucleus precipitating；The nucleus is deposited in second buffer and carries out the first resuspension processing, described
Two buffers first pass through 4 DEG C of precooling treatments in advance, and in first resuspension processing, re-suspension liquid is carried out pressure-vaccum 10 times repeatedly, institute
The dosage for stating the second buffer is 5mL；Processing product is resuspended by first and carries out second by the cell filter that diameter is 37 μm
Filtration treatment；The filtrate of second filtration treatment is carried out to the second centrifugal treating of 5min under conditions of 4 DEG C, 500g；By second
Precipitating after centrifugal treating carries out second and processing is resuspended and is carried out at third filtering by diameter for 37 μm of cell filter
Reason；Sucrose buffer I is added into the filtrate after third filtration treatment, pressure-vaccum mixes 10 times, and the Sucrose buffer I includes:
1.35ml Nuclei PURE 2M Sucrose buffer and 150 μ l Nuclei PURE Sucrose buffers；After pressure-vaccum is mixed 10 times
Filtrate be added to the upper layer the Sucrose buffer I, not mix, so as to obtain layering liquid；By the layering liquid temperature be 4
DEG C, speed be 13000g under conditions of centrifugal treating 45min；Product after centrifugal treating is removed into supernatant, it is described thin to obtain
Karyon, wherein first buffer includes: 8mM Tris alkali, the 0.8mM CaCl of 116.8mM NaCl, 7.8 pH2、38mM
MgCl2, 0.04%BSA, 0.16%Nonidet P-40,1mM EDTA, 1mg DAPI；Second buffer includes: 1 ×
PBS, 1.0%BSA and 0.2U/ μ L RNase inhibitor.Simple reagent consumptive material is only needed according to the method for the embodiment of the present invention,
Expensive separation equipment is not needed, and enough monokaryons can be obtained from less frost tumor tissues and surveyed for unicellular RNA
Sequence, and then nucleus suspension obtained is sequenced suitable for unicellular RNA according to the method for the embodiment of the present invention, can be applied to difficulty
To obtain the research such as flesh tissue, Tumor Heterogeneity that unicellular level can be carried out with the sample that frozen tissue replaces.
In the third aspect of the present invention, the invention proposes a kind of methods of unicellular sequencing.Implementation according to the present invention
Example, which comprises extract the nucleus in sample to be tested using mentioned-above method；The nucleus is carried out slender
Born of the same parents' transcript profile sequencing, to obtain sequencing result.As previously mentioned, a kind of method that inventor develops simplicity, it can be from frost
The monokaryon suspension for being suitable for unicellular sequencing is obtained in tumor tissues sample, relieves the unicellular sequencing clock synchronization of fresh tumor tissue
Between place limitation, and then unicellular sequencing technologies is allow to be applied more broadly in the research of tumor microenvironment.
According to an embodiment of the invention, the above method can further include at least one following additional technical feature:
According to an embodiment of the invention, before the nucleus carries out the unicellular transcript profile sequencing, it further will be described
Nucleus carries out resuspension processing, and after the resuspension processing, the concentration of nucleus is 1000 cores/μ L.Sequencing result is more quasi- as a result,
Really.
In another aspect of the invention, the method and/or extraction frost of the extraction nucleus of embodiment according to the present invention
Include the RNA of sufficient amount in the nucleus that the method for tumor tissue cell's core extracts, can be used for being sequenced, and with intact cell
It carries out RNA and correlation and consistency with higher is sequenced.
The method of the extraction nucleus of embodiment according to the present invention and/or the method for extracting frost tumor tissue cell's core
From frost tumor tissues separating nucleus carry out unicellular RNA sequencing can be applied at least one of in terms of:
1. tumour evolutionary mechanism is studied；
2. the accurate parting of cancer；
3. tumor drug resistance mechanism, outcome prediction are studied；
4. the research of tumor microenvironment, immunocyte and its relationship research with immunization therapy.
The method of the extraction nucleus of embodiment according to the present invention and/or the side for extracting frost tumor tissue cell's core
Method, compared with the existing technology in frost tumor tissue cell core separation method, there is at least one following advantage:
1) simplicity: only needing simple reagent consumptive material, does not need expensive separation equipment；
2) high efficiency: enough monokaryons can be obtained from less frost tumor tissues and are sequenced for unicellular RNA.
Detailed description of the invention
Fig. 1 is nucleus RNA clip size detection according to an embodiment of the present invention, it can be seen that the apparent peak 18S, 28S, table
Before bright RNA mass is better than optimization；
Fig. 2 is nucleus RNA segment detection before optimization according to an embodiment of the present invention, and RNA degradation is serious, the peak 18S, 28S
It is unobvious, RNA sequencing can not be carried out.
Specific embodiment
The embodiment of the present invention is described below in detail, examples of the embodiments are shown in the accompanying drawings.Below with reference to
The embodiment of attached drawing description is exemplary, it is intended to is used to explain the present invention, and is not considered as limiting the invention.
According to one embodiment of present invention, simply, efficiently divide from frost tumor tissues the invention proposes a kind of
Method from nucleus, gained monokaryon suspension are sequenced suitable for unicellular RNA, can be applied to be difficult to obtain flesh tissue, can be used
The sample of frozen tissue substitution carries out the research such as Tumor Heterogeneity of unicellular level.
A specific embodiment according to the present invention, the present invention is suitable for freezing the nucleus separation of tumor tissues, involved
And content mainly include 4 most of: preparation of reagents, histocyte cracking, nucleus cleaning, the detection of core suspension.
(1) preparation of reagents
1.NST lysis buffer (merges mixing after preparing following two components respectively):
1) 80mL NST:146mM NaCl, 10mM Tris base pH 7.8,1mM CaCl2,21mM MgCl2,
0.05%BSA, 0.2%Nonidet P-40)；
2) the 106mM MgCl of 20mL2, 5mM EDTA, 1mg DAPI
2.Nuclei Wash and Resuspension Buffer(Nuclei W&R Buffer):
1 × PBS, 1.0%BSA, 0.2U/ μ L RNase inhibitor.
(2) histocyte cracks
1.4 DEG C of pre-coolings: NST/DAPI lysis buffer, Nuclei W&R Buffer
It is pre-chilled on ice 2. plastic culture dish is placed on, 3mL NST/DAPI lysis buffer is added；
3. general~30mg tissue is put into culture dish, it is switched to the fragment less than 1mm3 with knife blade, places 15min on ice,
Every 5min jog mixes；
4. pressure-vaccum 10~15 times are broken up tissue, 15mL pipe is filled into 37 μm of cell filters；
(3) nucleus cleans
1. 4 DEG C of 500g of core suspension are centrifuged 5min；
2. carefully removing supernatant, core precipitating is not encountered；
3. the Nuclei W&R Buffer of 5mL pre-cooling, soft pressure-vaccum 10 times is added；
4. removing cell fragment and tissue mass twice with 37 μm of cell filter filterings；
5. 4 DEG C of 500g of core suspension are centrifuged 5min, supernatant is carefully removed, not encounter core precipitating；
6. 500 μ L Nuclei W&R Buffer are added, suspend completely to monokaryon for soft pressure-vaccum 10 times, again with cell mistake
Filter is filled into 1.5mL pipe；
(4) gradient centrifugation
Core suspension after cleaning is subjected to gradient centrifugation with sucrose cushion solution, the impurity such as removal cell fragment, purifying is carefully
Karyon.
(5) core suspension detects
With Countess II FL Automated Cell Counter automatic counter for counting detection nuclear concentration and cracking effect
Fruit.
Embodiment 1
(1) preparation of reagents
1.NST lysis buffer (merges mixing after preparing following two components respectively):
1) 80mL of NST:146mM NaCl, 10mM Tris base at pH 7.8,1mM CaCl2,21mM
MgCl2, 0.05%BSA, 0.2%Nonidet P-40)；
2) the 106mM MgCl of 20mL2, 5mM EDTA, 1mg DAPI；
2.Nuclei Wash and Resuspension Buffer(Nuclei W&R Buffer):
1 × PBS, 1.0%BSA, 0.2U/ μ L RNase inhibitor.
(2) histocyte cracks
1. prepared NST/DAPI lysis buffer, Nuclei W&R Buffer are pre-chilled in 4 DEG C of refrigerators；
It is pre-chilled on ice 2. plastic culture dish is placed on, 3mL NST/DAPI lysis buffer is added；
3. general~30mg tissue is put into culture dish, it is switched to the fragment less than 1mm3 with knife blade, places 15min on ice,
Every 5min jog mixes；
4. pressure-vaccum 10~15 times are broken up tissue, 15mL pipe is filled into 37 μm of cell filters；
(3) nucleus cleans
1. 4 DEG C of 500g of core suspension are centrifuged 5min；
2. carefully removing supernatant, core precipitating is not encountered；
3. the Nuclei W&R Buffer of 5mL pre-cooling, soft pressure-vaccum 10 times is added；
4. removing cell fragment and tissue mass twice with 37 μm of cell filter filterings；
5. 4 DEG C of 500g of core suspension are centrifuged 5min, supernatant is carefully removed, not encounter core precipitating；
6. 500 μ L Nuclei W&R Buffer are added, suspend completely to monokaryon for soft pressure-vaccum 10 times, again with cell mistake
Filter is filled into 1.5mL pipe；
(4) gradient centrifugation
1. preparing Sucrose buffer I:1.35ml Nuclei PURE 2M Sucrose buffer, 150 μ l Nuclei PURE are slow
Fliud flushing, it is ready-to-use, wherein Nuclei PURE 2M Sucrose buffer is purchased from SIGMA, and article No. is No.S9308, Nuclei
PURE buffer is purchased from SIGMA, and article No. is No.S9058.Wherein, Nuclei PURE 2M Sucrose buffer is containing 2M sucrose
Solution, Nuclei PURE buffer be for diluted buffer, this ratio mix it is resulting be 1.8M sucrose cushion
Liquid.M represents mol/L.
2. 900 μ l Sucrose buffer I are added in 500 μ l core suspensions, pressure-vaccum is mixed 10 times；
3. 500 μ l Sucrose buffer I are added in new 2mL pipe；
4. 1400 μ l core suspensions in step 2 to be carefully added into the upper layer of 500 μ l Sucrose buffer I, not mix；
5.4 DEG C of 13000g are centrifuged 45min；
6. carefully removing supernatant, nucleus is resuspended in residue~20 μ l solution, and suitable Nuclei W&R Buffer, which is added, to be made
Concentration about 1000nuclei/ μ l (1x 106nuclei/ml)；
(5) core suspension detects
1. 10 μ l core suspension Countess II FL Automated Cell Counter automatic counter for counting is taken to detect core
Concentration and lytic effect.Concentration, lytic effect detection, it is known that, total cell concentration 4.40*105/ mL, wherein viable cell concentrations
For 1.17*104/ mL accounts for 3%, and dead cell concentration is 4.28*105/ mL, accounts for 97%, shows to have obtained the nucleus of single suspension,
Cell debris, cleavage rate 97% meet the requirement of the unicellular sequencing experiment of 10X Genomics.Detection hair is dyed by DAPI
It is existing, it is 9.00*10 in total cell concentration4Under conditions of/mL, there is 5.37*104/ mL is that cell is colored, i.e., rate of dyeing is 60%.
2. taking about 12000 monokaryons to use the unicellular microarray dataset Chromium of 10X Genomics according to nuclear concentration
3 ' Solution v2 reagent of Controller and Chromium Single Cell carries out unicellular 3 ' RNA sequencing library structure
It builds, Illumina HiSeq sequencer, 10X Genomics Cell Ranger Data Analysis Software carries out data analysis
3. detecting RNA segment with Agilent 2100Bioanalyzer, as a result seeing attached remaining nucleus extraction RNA
Fig. 1.It can be seen that the apparent peak 18S, 28S, before showing RNA mass better than optimization.
Comparative example 1 (is not added with BSA in Nuclei W&R Buffer)
1. preparing buffer, 4 DEG C of pre-coolings:
NST lysis buffer:800mL of NST (146mM NaCl, 10mM Tris base at pH 7.8,1mM
CaCl2,21mM MgCl2, 0.05%BSA, 0.2%Nonidet P-40), 200mL of 106mM MgCl2, 5mM EDTA,
1mg DAPI,
Nuclei Wash and Resuspension Buffer (Nuclei W&R Buffer): 0.35 × PBS；
It is pre-chilled on ice 2. plastic culture dish is placed on, 1mL NST lysis buffer is added；
3. tissue is put into culture dish, shredded with knife blade；
4. core suspension is obtained by filtration with 37 μm of cell filters；
5. being diluted with 0.35 × PBS, Countess II FL Automated Cell Counter is counted, and cell is always dense
Degree is 7.04*104/ mL, wherein viable cell concentrations are 6.45*104/ mL accounts for 92%, and dead cell concentration is 5.86*103/ mL, accounts for
8%, it can be seen that nuclear concentration is lower, and lytic effect is poor, is unsatisfactory for unicellular sequencing requirement of experiment.
6. detecting RNA segment with Agilent 2100Bioanalyzer, as a result seeing attached remaining nucleus extraction RNA
Fig. 2.RNA degradation is serious, can not carry out RNA sequencing.
Comparative example 2 (not plus gradient centrifugation step)
1. preparing buffer, 4 DEG C of pre-coolings:
NST lysis buffer:800mL of NST (146mM NaCl, 10mM Tris base at pH 7.8,1mM
CaCl2,21mM MgCl2,0.05%BSA, 0.2%Nonidet P-40), 200mL of 106mM MgCl2,5mM EDTA,
1mg DAPI,
Nuclei Wash and Resuspension Buffer (Nuclei W&R Buffer): 1 × PBS, 1.0%
BSA
It is pre-chilled on ice 2. plastic culture dish is placed on, 3mL NST lysis buffer is added；
3. general~30mg tissue is put into culture dish, with knife blade chopping to less than the fragment of 1mm3, place on ice
15min；
4. pressure-vaccum 10~15 times are broken up tissue, 15mL pipe is filled into 37 μm of cell filters；
5. 4 DEG C of 500g of core suspension are centrifuged 5min；
6. carefully removing supernatant, core precipitating is not encountered；
7. the Nuclei W&R Buffer of 5mL pre-cooling, soft pressure-vaccum 10 times is added；
8. removal cell fragment and tissue mass is filtered for multiple times with 37 μm of cell filters；
9. 4 DEG C of 500g of core suspension are centrifuged 5min, supernatant is carefully removed, not encounter core precipitating；
10. 200 μ L Nuclei W&R Buffer are added, suspend completely to monokaryon for soft pressure-vaccum 10 times, uses cell again
Filter is filled into 1.5mL pipe；
Countess II FL Automated Cell Counter is counted, total cell concentration 9.26*106/ mL,
In lytic cell concentration be 8.38*106/ mL accounts for 90%, and nuclear concentration is higher, but there are more cell fragments.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means specific features, structure, material or spy described in conjunction with this embodiment or example
Point is included at least one embodiment or example of the invention.In the present specification, schematic expression of the above terms are not
It must be directed to identical embodiment or example.Moreover, particular features, structures, materials, or characteristics described can be in office
It can be combined in any suitable manner in one or more embodiment or examples.In addition, without conflicting with each other, the skill of this field
Art personnel can tie the feature of different embodiments or examples described in this specification and different embodiments or examples
It closes and combines.
Although the embodiments of the present invention has been shown and described above, it is to be understood that above-described embodiment is example
Property, it is not considered as limiting the invention, those skilled in the art within the scope of the invention can be to above-mentioned
Embodiment is changed, modifies, replacement and variant.
Claims (10)
1. a kind of method for extracting nucleus, which is characterized in that
Sample to be extracted is subjected to cracking processing, the cracking processing is carried out in the first buffer；
Pyrolysis product is subjected to the first centrifugal treating, to obtain nucleus precipitating；
Nucleus precipitating is rinsed and resuspension is handled, rinses and resuspension processing is carried out in the second buffer,
To obtain the nucleus；
Wherein, first buffer includes: 8mM Tris alkali, the 0.8mM CaCl of 116.8mM NaCl, 7.8 pH2、38mM
MgCl2, 0.04%BSA, 0.16%Nonidet P-40,1mM EDTA, 1mg/mL DAPI；
Second buffer includes: 1 × PBS, 1.0%BSA and 0.2U/ μ L RNase inhibitor.
2. the method according to claim 1, wherein first buffer and the second buffer first pass through in advance
4 DEG C of precooling treatments；
Optionally, the sample to be extracted is frost tumor tissues, the quality of the sample to be extracted and first buffer
Volume ratio is 30mg:3mL.
3. according to the method described in claim 2, it is characterized in that, cracking processing carries out in the following way:
The sample to be extracted is subjected to Mechanical Crushing processing in first buffer, the sample size after break process is not
Greater than 1mm3；
Mechanical Crushing processing product ice is set into processing 30min, every 5min is mixed；
Ice is set into processing product and carries out 10~15 piping and druming processing, and piping and druming processing product is subjected to the first filtration treatment,
First filtration treatment is that the cell filter for being 37 μm by diameter carries out, the filtrate structure after first filtration treatment
At the pyrolysis product.
4. the method according to claim 1, wherein first centrifugal treating is under conditions of 4 DEG C, 500g
Carry out 5min.
5. the method according to claim 1, wherein the flushing and resuspension processing are to carry out in the following way
:
The nucleus is deposited in second buffer and carries out the first resuspension processing, the dosage of second buffer is
5mL；
Processing product is resuspended by first and carries out the second filtration treatment；
The filtrate of second filtration treatment is subjected to the second centrifugal treating, second centrifugal treating is under conditions of 4 DEG C, 500g
Carry out 5min；
Precipitating after second centrifugal treating is carried out second, processing and third filtration treatment is resuspended；
Preferably, in first resuspension processing, re-suspension liquid is subjected to pressure-vaccum 10 times repeatedly；
Preferably, second, third described filtration treatment is that the cell filter for being 37 μm by diameter carries out.
6. according to the method described in claim 5, it is characterized in that, further comprising by the after the flushing and resuspension processing
Filtrate after three filtration treatments carries out gradient centrifugation processing.
7. according to the method described in claim 6, it is characterized in that, the gradient centrifugation is carried out in Sucrose buffer I；
Optionally, the Sucrose buffer I includes: 1.35ml Nuclei PURE 2M Sucrose buffer and 150 μ l Nuclei
PURE buffer；
Optionally, the gradient centrifugation carries out in the following way:
The Sucrose buffer I is added into the filtrate after the third filtration treatment, pressure-vaccum mixes 10 times；
Filtrate after pressure-vaccum is mixed 10 times is added to the upper layer the Sucrose buffer I, not mix, to obtain layering liquid；
By the layering liquid under conditions of temperature is 4 DEG C, speed is 13000g centrifugal treating 45min；
Product after centrifugal treating is removed into supernatant, to obtain the nucleus.
8. a kind of method for extracting frost tumor tissue cell's core characterized by comprising
Frost tumor tissues are subjected to Mechanical Crushing processing in the first buffer, the sample size after break process is not more than
1mm3；First buffer first passes through 4 DEG C of precooling treatments, the quality of the frost tumor tissues and first buffer in advance
Volume ratio is 30mg:3mL；
Mechanical Crushing processing product ice is set into processing 30min, every 5min is mixed；
Ice is set into processing product and carries out 10~15 piping and druming processing, and piping and druming processing product is subjected to the first filtration treatment,
First filtration treatment is that the cell filter for being 37 μm by diameter carries out, the filtrate structure after first filtration treatment
At pyrolysis product；
Pyrolysis product is subjected to the first centrifugal treating, first centrifugal treating is to carry out 5min under conditions of 4 DEG C, 500g,
To obtain nucleus precipitating；
The nucleus is deposited in second buffer and carries out the first resuspension processing, second buffer first passes through in advance
Re-suspension liquid is carried out pressure-vaccum 10 times repeatedly, the use of second buffer in first resuspension processing by 4 DEG C of precooling treatments
Amount is 5mL；
Processing product is resuspended by first, second filtration treatment is carried out by the cell filter that diameter is 37 μm；
The filtrate of second filtration treatment is carried out to the second centrifugal treating of 5min under conditions of 4 DEG C, 500g；
Precipitating after second centrifugal treating is subjected to the second resuspension processing and is carried out by diameter for 37 μm of cell filter
Third filtration treatment；
Sucrose buffer I is added into the filtrate after third filtration treatment, pressure-vaccum mixes 10 times, and the Sucrose buffer I includes:
1.35ml Nuclei PURE 2M Sucrose buffer and 150 μ l Nuclei PURE buffers；
Filtrate after pressure-vaccum is mixed 10 times is added to the upper layer the Sucrose buffer I, not mix, to obtain layering liquid；
By the layering liquid under conditions of temperature is 4 DEG C, speed is 13000g centrifugal treating 45min；
Product after centrifugal treating is removed into supernatant, to obtain the nucleus,
Wherein, first buffer includes: 8mM Tris alkali, the 0.8mM CaCl of 116.8mM NaCl, 7.8 pH2、38mM
MgCl2, 0.04%BSA, 0.16%Nonidet P-40,1mM EDTA, 1mg/mL DAPI；
Second buffer includes: 1 × PBS, 1.0%BSA and 0.2U/ μ L RNase inhibitor.
9. a kind of method of unicellular sequencing, which is characterized in that using according to any one of claims 1 to 88 described in any item methods extract to
Nucleus in sample；
Unicellular transcript profile sequencing is carried out to the nucleus, to obtain sequencing result.
10. according to the method described in claim 9, it is characterized in that, the nucleus carries out the unicellular transcript profile sequencing
Before, the nucleus is further subjected to resuspension processing, after the resuspension processing, the concentration of nucleus is 1000 cores/μ L.
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