CN111501106A

CN111501106A - Construction method, device and application of high-throughput sequencing library of exosome RNA

Info

Publication number: CN111501106A
Application number: CN202010336574.1A
Authority: CN
Inventors: 杨玲; 管彦芳; 张燕艳; 姬利延; 贾明玺
Original assignee: Beijing Jiyinjia Medical Laboratory Co ltd; Beijing Jiyingjia Technology Co ltd
Current assignee: Beijing Jiyinjia Medical Laboratory Co ltd; Beijing Jiyingjia Technology Co ltd
Priority date: 2020-04-24
Filing date: 2020-04-24
Publication date: 2020-08-07

Abstract

The invention discloses a construction method of a high-throughput sequencing library of exosome RNA, which comprises the following steps: extracting total RNA of exosome; fragmenting the total RNA to obtain fragmented RNA; reverse transcribing the fragmented RNA into double-stranded cDNA; connecting a sequencing linker at the 5 'end and the 3' end of the double-stranded cDNA to obtain a linker connection product; amplifying the adaptor connection product by PCR to obtain an enriched adaptor connection product; and separating the single-stranded DNA from the enriched adaptor ligation product, and performing cyclization treatment on the single-stranded DNA to obtain circular DNA, wherein the circular DNA forms a high-throughput sequencing library of the exosome RNA. The construction method can realize the construction of a sequencing library of the exosome RNA with low initial quantity, the sequencing result widely covers various RNAs such as mRNA, miRNA, lncRNA and the like, and the construction method has important values in the aspects of early diagnosis, prognosis evaluation, curative effect monitoring, treatment method research and the like of diseases.

Description

Construction method, device and application of high-throughput sequencing library of exosome RNA

Technical Field

The invention relates to the technical field of high-throughput sequencing, in particular to a construction method, a device and application of a high-throughput sequencing library of exosome RNA.

Background

Exosomes (exosomes) are a class of closed membrane vesicles of lipid bilayers with a diameter of 30-150nm and a density of 1.10-1.21ng/ml, with cytoplasmic and membrane components of the originating cell. Exosomes were first observed during maturation of reticulocytes from mature mammals, and later studies found that most cell lines, such as mast cells, dendritic cells, lymphocytes, fibroblasts, mesenchymal stem cells, and tumor cells, can produce and release exosomes. Exosomes, which are Multivesicular Vesicles (MVBs) derived from endosomes, are widely involved in various cell pathophysiological processes. The exosome is wrapped with a plurality of components such as protein, nucleic acid, lipid and the like, wherein RNA (mRNA, ncRNA) is enriched in the exosome, has good stability under the protection of the surface membrane of the exosome, and is an ideal biomolecule reflecting a specific physiological or pathological state.

The transcriptome sequencing (RNA-Seq) technology is an important tool for deeply researching RNA function and expression change, and can detect the overall transcription activity of any species at a single nucleotide level. Currently, in the research of analyzing exosome RNA by RNA-Seq, a corresponding mRNA sequencing library and a corresponding miRNA sequencing library are mainly constructed based on mRNA and miRNA. In constructing the sequencing library, it is necessary to use oligo (dT) capture technology or design probes to specifically remove ribosomal RNA (rRNA) from total RNA of exosomes. However, the RNA Integrity Number (RIN) of the exosome is in the range of 1-3 and is low in content. The existing construction method of exosome RNA sequencing library causes great loss of RNA quantity, and usually requires a high initial quantity of RNA (at least >10ng) to meet the requirement of library construction; in addition, due to the influence of RNA loss in the library construction process, the RNA-Seq is difficult to widely cover various RNAs in exosomes, and the application of exosome RNA in clinical diagnosis, biomedical research and other aspects is limited.

Disclosure of Invention

Therefore, the technical problem to be solved by the invention is to overcome the defects that the construction of an exosome RNA sequencing library in the prior art needs high RNA initial amount and is difficult to widely cover each exosome RNA.

Therefore, the invention provides the following technical scheme:

in a first aspect, the invention provides a method for constructing a high-throughput sequencing library of exosome RNA, which comprises the following steps:

s1, extracting total RNA of the exosome;

s2, fragmenting the total RNA to obtain fragmented RNA;

s3, reverse transcribing the fragmented RNA into double-stranded cDNA;

s4, connecting sequencing adapters at the 5 'end and the 3' end of the double-stranded cDNA to obtain adapter connection products; PCR amplifying the adaptor-ligated product to obtain an enriched adaptor-ligated product;

s5, separating single-stranded DNA from the enriched adaptor ligation product, and performing cyclization treatment on the single-stranded DNA to obtain circular DNA, wherein the circular DNA forms a high-throughput sequencing library of the exosome RNA.

Optionally, in the above construction method, the step S3 includes:

s31, reverse transcribing the fragmented RNA into double-stranded cDNA;

s32, purifying the double-stranded cDNA by using magnetic beads to obtain purified double-stranded cDNA;

s33, performing end repair on the purified double-stranded cDNA, and adding bases A to the 5 'end and the 3' end of the purified double-stranded cDNA to obtain end-repaired double-stranded cDNA;

optionally, in the above construction method, the step S4 includes:

s41, connecting sequencing adapters at the 5 'end and the 3' end of the double-stranded cDNA to obtain adapter connection products;

s42, purifying the adaptor connection product by using magnetic beads to obtain a purified adaptor connection product;

s43, carrying out PCR amplification on the purified adaptor connection product to obtain the enriched adaptor connection product;

s44, purifying the enriched adaptor connection product by magnetic beads to obtain the adaptor connection product suitable for separating single-stranded DNA.

Optionally, in the above construction method, the total RNA comprises: mRNA, miRNA, lncRNA, snRNA, snoRNA.

Optionally, in the above construction method, the sequencing linker is a Y-linker formed by partially complementing a first sequence and a second sequence, the first sequence is a single-stranded nucleotide with a nucleotide sequence shown in SEQ ID No.1, and the second sequence is a single-stranded nucleotide with a nucleotide sequence shown in SEQ ID No. 2;

the primers for PCR amplification comprise: forward primer, the nucleotide sequence of which is shown in SEQ ID NO. 3; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 4.

Alternatively, the above-described construction method,

the Reaction solution for fragmenting the total RNA comprises 5 mu L of the total RNA, 4 mu L of NEB Next First Strand Synthesis Reaction Buffer, 1 mu L of Random Primers;

the reaction program for the total RNA fragmentation treatment is as follows: reacting at 94 ℃ for 7 min;

reverse transcription of the fragmented RNA into double-stranded cDNA comprises a first strand synthesis reaction and a second strand synthesis reaction;

the reaction system of the First Strand Synthesis reaction comprises a reaction solution containing fragmented RNA, 10 mu L, nucleic-free Water, 8 mu L, NEB Next First Strand and Synthesis Enzyme Mix, 2 mu L;

the reaction sequence of the first strand synthesis reaction includes: reacting at 25 deg.C for 10min, at 42 deg.C for 15min, at 70 deg.C for 15min, and maintaining at 4 deg.C;

the Reaction system of the Second Strand Synthesis Reaction comprises a Reaction solution containing a first Strand, 20 mu L, NEB New Second Strand and Synthesis Reaction Buffer (10X), 8 mu L, NEB New Second Strand and Synthesis Enzyme Mix, 4 mu L, nucleic-free Water, 48 mu L;

the reaction sequence of the second strand synthesis reaction includes: reacting at 16 ℃ for 1 h.

Optionally, in the above construction method, the Reaction solution for End repair of double-stranded cDNA comprises a Reaction solution containing double-stranded cDNA, 50. mu. L, NEB Next Ultra II End Prep Reaction Buffer, 7. mu. L, NEB Nextultra II End Prep Enzyme Mix, 3. mu. L;

the reaction program for performing end repair on the double-stranded cDNA comprises the following steps: reacting at 20 ℃ for 30min and at 65 ℃ for 30 min;

the reaction solution for connecting the double-stranded cDNA with the 5 'joint and the 3' joint comprises a reaction solution containing double-stranded cDNA with the end repaired, 60 mu L, NEB Next L alignment Enhancer, 1 mu L, NEB Next Ultra II L alignment Master Mix, 30 mu L, 5 'joint and 3' joint, 1.5 mu M and 2 mu L;

the reaction procedure for the double-stranded cDNA ligation of the 5 'adaptor and the 3' adaptor comprises: incubating at 20 deg.C for 15 min;

the reaction solution for enriching the adaptor ligation product comprises Universal PCR Primer/i5Primer, 10 mu M, 2.5 mu L, index (X) Primer/i7Primer, 10 mu M, 2.5 mu L, NEB Next Ultra II Q5Master Mix, 25 mu L, reaction solution containing the adaptor ligation product, 20 mu L;

the reaction procedure for enriching the linker ligation product comprises: pre-denaturation at 98 ℃ for 30 s; denaturation at 98 ℃ for 10s, annealing at 65 ℃ for 75s, and 7-16 cycles; extending for 5min at 65 ℃; keeping at 4 ℃.

In a second aspect, the present invention provides a high throughput sequencing library of exosome RNAs, the sequencing library being obtained based on the construction method of any one of claims 1 to 7.

In a third aspect, the present invention provides an apparatus for constructing a high throughput sequencing library of exosome RNAs, comprising:

an extraction module for extracting total RNA of exosomes;

a fragmentation processing module, configured to perform fragmentation processing on the total RNA to obtain fragmented RNA;

a reverse transcription module for reverse transcribing the fragmented RNA into double-stranded cDNA;

a linker connection module for connecting a sequencing linker at the 5 'end and the 3' end of the double-stranded cDNA to obtain a linker connection product; PCR amplifying the adaptor-ligated product to obtain an enriched adaptor-ligated product;

a circularization module for isolating single stranded DNA from the enriched adaptor ligation products, subjecting the single stranded DNA to circularization to obtain circular DNA constituting a high throughput sequencing library of the exosome RNA.

In a fourth aspect, the invention provides the use of any one of the following a1-a 6:

a1, the use of the above construction method in exosome RNA sequencing;

a2, the use of the above construction method in constructing an exosome RNA sequencing library;

a3, use of the high throughput sequencing library described above in exosome RNA sequencing;

a4, use of the device described above in exosome RNA sequencing;

a5, the use of the device described above for constructing an exosome RNA sequencing library;

a6, and the application of the construction method, the high-throughput sequencing library or the device in preparing products for early disease screening, prognosis evaluation and/or clinical efficacy monitoring.

The technical scheme of the invention has the following advantages:

according to the construction method of the high-throughput sequencing library of the exosome RNA, provided by the invention, the library construction process is optimized, the RNA sequencing library is constructed based on the total RNA of exosomes, the rRNA does not need to be removed, the RNA integrity in exosomes is favorably maintained, the loss of RNA content and types in the library construction process is reduced, the final RNA sequencing can cover multiple RNA types such as mRNA, miRNA, lncRNA, snRNA and snoRNA, and the exosome detection of a high-coverage RNA spectrum is realized. In addition, the total RNA is used as a template for reverse transcription in the library building process, sequencing joints are added at the 5 'end and the 3' end of the cDNA simultaneously, and the joint connection products are subjected to single-strand cyclization to obtain the RNA sequencing library of the single-strand circular DNA, so that the requirement of multi-platform sequencing can be met, and the high efficiency and accuracy of the sequencing result can be ensured. The construction method is simple to operate and good in effect, and can meet the library construction requirement of low-quality and low-content exosome RNA.

The construction method of the high-throughput sequencing library of the exosome RNA provided by the invention can realize the construction of the sequencing library of the exosome RNA with low initial amount (as low as 1ng) by further optimizing the reaction program and the reaction system, lays a foundation for differential expression analysis, expression profile analysis, prediction and identification of disease associated genes and the like of the exosome RNA, and has important application value in the aspects of early diagnosis, prognosis evaluation, curative effect monitoring, research on treatment methods and the like.

According to the construction method of the high-throughput sequencing library of the exosome RNA, provided by the invention, the two ends of the double-stranded cDNA fragment are connected with the Y-shaped joints formed by partially complementing the first sequence and the second sequence, the joint connection efficiency of the double-stranded cDNA fragment is high, different nucleotide sequences of the first sequence and the second sequence are introduced into the two ends of a connection product after PCR amplification, and the construction of the low-initial-amount exosome RNA sequencing library and subsequent high-throughput sequencing detection are favorably realized.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 shows a quality control diagram of high throughput sequencing library 2100 for exosome RNA in example 1 of the present invention;

FIG. 2 shows a high throughput sequencing library L abChip peak profile for exosome RNAs in example 1 of the present invention;

FIG. 3 shows the ratio of different RNA types in the RNA sequencing of exosomes according to the invention in Experimental example 1.

Detailed Description

The technical solutions of the present invention will be described clearly and completely below, and it should be apparent that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. In addition, the technical features involved in the different embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

The experimental reagents used in the following examples are all conventional experimental reagents, and are directly available from the market;

example 1

The embodiment provides a construction method of a high-throughput sequencing library of exosome RNA, which comprises the following steps:

a method for constructing a high-throughput sequencing library of exosome RNA,

s1, extracting total RNA of the exosome, which comprises the following steps:

(1) centrifuging the thawed sample at 4 deg.C (3000x g 15min/16000x g 10min), adding centrifuged supernatant (2-4ml) into a 15ml centrifuge tube with corresponding number, and covering with a cover;

(2) adding equal volume of Buffer XBP into the centrifuge tube, covering the centrifuge tube with a cover, and slightly turning upside down for several times to fully and uniformly mix the mixture;

(3) adding the sample and Buffer XBP mixed solution into exo easy spin column, centrifuging for 1min at 500x g, discarding the effluent, and placing the centrifugal column back into the corresponding collection tube;

(4) adding 10ml XWP into the centrifugal column, covering the centrifugal column with a cover, centrifuging for 5min at 5000x g, and discarding an effluent liquid and a collecting pipe; preparing a new collecting pipe, marking the sample number, and placing the centrifugal column in the corresponding collecting pipe;

(5) adding 700ul QIAzol into the middle of the silica gel membrane, centrifuging for 5min at 5000x g, collecting lysate, and completely transferring into a 2ml centrifuge tube;

(6) vortex the centrifuge tube and incubate for 5min at room temperature;

(7) adding 90 mul of chloroform into the centrifuge tube, covering the centrifuge tube with a cover, and performing vortex oscillation for 15 s;

(8) incubating at room temperature for 3 min;

(9) centrifuging for 15min at 4 ℃ of 12000x g by using a standard centrifuge;

(10) transferring the upper colorless aqueous phase into a 1.5ml centrifuge tube to avoid contacting the intermediate interface, adding 2 times of 100% absolute ethyl alcohol (about 800 μ l) into the centrifuge tube, mixing uniformly without centrifugation, and immediately performing the next step;

(11) adding 700ul of the mixed solution (including the precipitate which may be generated) into RNeasy MinElute spin column, covering the lid, centrifuging with a standard centrifuge of 8000 or more 8000x g (10,000 rpm) for 15s, and discarding the effluent;

(12) adding 700ul Buffer RWT to a MinElute spin column, covering a cover, centrifuging at 8000x g (10,000 rpm) for 15s, and discarding the effluent;

(13) adding 500ul Buffer RPE into MinElute spin column, covering with cover, centrifuging at 8000x g (10,000 rpm) for 15s, and discarding the effluent;

(14) adding 500ul Buffer RPE into MinElute spin column, covering with cover, centrifuging at 8000x g (10,000 rpm) for 2min, and removing effluent and collection tube;

(15) placing the MinElute spin column in a new 2ml collection tube, centrifuging at full speed for 2min, discarding the collection tube, opening the lid of the MinElute spin column, and standing on an ultra-clean bench for 5min (thoroughly drying);

(16) putting a MinElute spin column into a 1.5ml collecting tube, adding 14ul of nuclease-free water into the middle of the membrane, covering a cover, standing and incubating for 5min, centrifuging at full speed for 2min to elute RNA, removing the spin column, covering a tube cover for collecting RNA, attaching a corresponding sample label, and performing RNA quality control detection.

(17) exosomal RNA extracted from exoRNeasy Serum/Plasma Maxi Kit.

S2, fragmenting the total RNA to obtain fragmented RNA; specifically, the method comprises the following steps:

(1) starting an ice maker to make ice in advance, opening a superclean workbench, and sterilizing for 30min by ultraviolet rays;

(2) preparing corresponding quantities of 0.2ml centrifuge tubes without RNAase and gun heads according to the database building task list, and marking sample numbers on tube covers;

(3) taking out NEBNext First Strand Synthesis Reaction Buffer and RandomPrimers in advance, placing on ice, fully oscillating, uniformly mixing and centrifuging after dissolving;

(4) the Premix of the fragmentation reaction was formulated on ice as in table 1 below:

TABLE 1

(5) Fully oscillating, uniformly mixing and centrifuging;

(6) subpackaging Premix, reacting 5ul each time, sequentially adding 5ul of RNA sample, shaking, mixing uniformly and centrifuging;

(7) reacting for 7min at 94 ℃ on a PCR instrument; fragmented exosome RNA was obtained by high temperature disruption.

S3, reverse transcribing the fragmented RNA into double-stranded cDNA; the method comprises the following steps:

s31, reverse transcribing the fragmented RNA into double-stranded cDNA; the method specifically comprises the following steps:

(1) taking out one-chain synthetic reaction solution (such as NEBNext Strand Specificity Reagent) in advance, placing on ice, dissolving, shaking thoroughly, mixing, and centrifuging with one-chain synthetase (such as NEBNext First Strand Synthesis enzymeMix);

(2) the Premix for the first chain reaction was synthesized as follows:

TABLE 2

(3) Fully oscillating, uniformly mixing and centrifuging;

(4) subpackaging Premix, reacting 10ul each time, sequentially adding 10ul of reaction solution, oscillating, mixing uniformly and centrifuging;

(5) the PCR reaction program (hot lid temperature greater than 80 ℃) was set up as in Table 3 below:

TABLE 3

Temperature of	25℃	42℃	70℃	4℃
					Time of day	10min	15min	15min	∞

(6) Taking out the reaction solution for two-chain Synthesis (such as NEBNext Second Strand and Synthesis reaction buffer), dUTP and Enzyme-free water, placing on ice, dissolving, shaking thoroughly, mixing, and centrifuging with two-chain synthetase (such as NEBNext Second Strand and Synthesis Enzyme Mix);

(7) the Premix for the second chain reaction was prepared on ice according to the following table 4:

TABLE 4

(8) Fully oscillating, uniformly mixing and centrifuging;

(9) subpackaging the premixed solution, reacting 60ul each time, sequentially adding 20ul of the reaction solution, oscillating, uniformly mixing and centrifuging;

(10) reacting for 1h at 16 ℃ on a PCR instrument (the temperature of a hot cover is less than 40 ℃);

s32, purifying the double-stranded cDNA by using magnetic beads to obtain purified double-stranded cDNA; the method specifically comprises the following steps:

(1) after the double-stranded cDNA was synthesized, the cDNA was purified using magnetic beads, which were allowed to equilibrate at room temperature for 30min before use.

(2) Adding 144ul (1.8X) AMpure XP magnetic beads into each reaction tube, blowing or slightly shaking and mixing uniformly, and then incubating for 10min at room temperature to ensure that the magnetic beads are fully combined with the cDNA fragments;

(3) after slight centrifugation, the centrifuge tube was placed on a magnetic rack until the solution was clear;

(4) adding 200 mul of 80% ethanol to wash twice, and incubating for 30s each time;

(5) completely removing ethanol, opening a cover, heating and drying the magnetic beads by a 37 ℃ dry heater until the magnetic beads do not shine or crack;

(6) adding 53 μ l of 0.1 XTE buffer solution into the centrifuge tube in sequence, sucking and pumping by a pipette gun, mixing uniformly, and incubating at room temperature for 5 min;

(7) placing the centrifugal tube on a magnetic frame after instantaneous centrifugation until the centrifugal tube is completely clear;

(8) and transferring the supernatant purified product into a new centrifuge tube, discarding the magnetic bead tube, and corresponding tube labels one by one during transfer to avoid confusion.

(1) opening two constant temperature blending instruments in advance, setting the temperature at 20 ℃ and 65 ℃ respectively, and setting the time at 30 min;

(2) taking out the End repairing reaction solution (such as NEBNext Ultra II End Prep reaction buffer) in advance, placing on ice, fully shaking and mixing uniformly after dissolving, and centrifuging with End repairing enzyme (such as NEBNext Ultra II End PrepEnzyme Mix);

(3) a premix of tip repair and addition of "a" was prepared on ice as in table 5 below:

TABLE 5

(4) Fully oscillating, uniformly mixing and centrifuging;

(5) subpackaging the premixed solution, reacting 10ul each time, sequentially adding 50ul of the reaction solution, oscillating, uniformly mixing and centrifuging;

(6) incubate on a constant temperature mixer as per the following table 6 requirements:

TABLE 6

Temperature of	20℃	65℃
			Time of day	30min	30min

(7) After the incubation is completed, the incubation tube is centrifuged instantaneously, and the evaporated liquid is collected in the tube.

S41, connecting sequencing adapters at the 5 'end and the 3' end of the double-stranded cDNA to obtain adapter connection products; specifically, the method comprises the following steps:

(1) opening the constant temperature blending instrument in advance, setting the temperature to be 20 ℃ and setting the time to be 15 min;

(2) the ahead dilution joints were required according to table 7 below, the joint diluent had to be cryogenic, and the dilution process operated on ice:

TABLE 7

Initial concentration of linker	Dilution factor
		15uM	Diluting the joint diluent by 10 times

The sequencing joint is a Y-shaped joint formed by partially complementing a first sequence and a second sequence, wherein the first sequence is a single-stranded nucleotide with a nucleotide sequence shown as SEQ ID NO.1, and the second sequence is a single-stranded nucleotide with a nucleotide sequence shown as SEQ ID NO. 2.

(3) The DNA ligase and ligase reaction buffer (such as NEB Next L alignment Enhancer and NEB Next Ultra II L alignment Master Mix) were removed in advance, mixed, centrifuged, placed on ice, and the adaptor reaction premix was prepared as required in Table 8 below:

TABLE 8

(4) Fully oscillating, uniformly mixing and centrifuging;

(5) subpackaging Premix, reacting 31ul, sequentially adding 60ul of reaction solution, adding 2ul of diluted joint into each tube, oscillating, mixing uniformly and centrifuging;

(6) incubate at 20 ℃ for 15min on a constant temperature mixer.

S42, purifying the adaptor connection product by using magnetic beads to obtain a purified adaptor connection product; specifically, the method comprises the following steps:

(1) and after the connection reaction is finished, purifying the joint connection product by using magnetic beads, balancing the magnetic beads at room temperature for 30min before use, opening a heat drier in advance, and setting the temperature to be 37 ℃.

(2) Adding 87ul (0.9X) AMpure XP magnetic beads into each reaction tube, blowing or slightly shaking and uniformly mixing, and incubating at room temperature for 10min to ensure that the magnetic beads are fully combined with the DNA fragments;

(4) discarding the supernatant, opening one centrifugal tube each time in order to reduce the pollution risk among samples, closing a tube cover after removing the supernatant, and opening the other centrifugal tube;

(5) adding 200ul 80% ethanol, washing twice, and incubating for 30s each time;

(6) completely removing ethanol, opening a cover, placing the centrifuge tube in a 37 ℃ dry heater for heating and drying until the surfaces of the magnetic beads are not reflected, taking down the centrifuge tube from the dry heater, and covering the tube cover;

(7) adding 17ul of 0.1 XTE buffer solution into the centrifuge tube in sequence, sucking and pumping the buffer solution by a pipette gun, mixing the buffer solution uniformly, and incubating the mixture for 5min at room temperature;

(8) placing the centrifugal tube on a magnetic frame after instantaneous centrifugation until the centrifugal tube is completely clear;

(9) transferring the supernatant purified product into a new centrifuge tube, discarding the magnetic bead tube, and corresponding tube labels one by one during transfer to avoid confusion;

s43, carrying out PCR amplification enrichment on the purified adaptor connection product to obtain the adaptor connection product suitable for separating single-stranded DNA. Specifically, the method comprises the following steps:

(1) the PCR polymerase mixture (e.g., NEBNext Ultra II Q5Master Mix) was removed, mixed and centrifuged, placed on ice, the reaction components added to the PCR tubes in the order of Table 9 below, and the negative/positive controls set;

TABLE 9

(2) Oscillating, mixing evenly and centrifuging;

(3) the corresponding relationship of the number of cycles of the PCR on-board sample is shown in Table 10:

watch 10

Initial amount of RNA	Number of cycles
		1000ng	7-8
100ng	11-12
		10ng	14-15
5ng	15-16

The PCR programming is shown in table 11 below:

TABLE 11

S44, purifying the enriched adaptor connection product by magnetic beads to obtain the adaptor connection product suitable for separating single-stranded DNA. Specifically, the method comprises the following steps:

(1) the PCR product was purified using 45ul (0.9X) AMpure XP magnetic beads and finally dissolved in 20ul TE (pH 8);

(2) quantifying the purified product Qubit-BR;

(3) library quality control (e.g., L abchip or 2100 bioanalyzer) is performed and qualified libraries are subjected to the circularization pooling step.

S5, separating single-stranded DNA from the joint connection product, and carrying out cyclization treatment on the single-stranded DNA to obtain circular DNA, wherein the circular DNA forms a high-throughput sequencing library of the exosome RNA. Specifically, the method comprises the following steps:

(1) single strand cyclization

1) Based on the length of the insert of library DNA, 1pmol was taken into a 0.2m L PCR tube and supplemented with TE Buffer to a total volume of 48. mu. L.

2) Placing the PCR tube in the step (1) on a PCR instrument, and carrying out reaction according to the conditions in the following table 12:

TABLE 12

Temperature of	Time of day
		Hot lid	On
95℃	3min

3) After the reaction was completed, the PCR tube was immediately transferred to ice and left for 2 min.

(2) Single strand cyclization

1) Single-chain cyclization reactions were prepared on ice as follows in Table 13:

watch 13

2) The 12.1. mu. L single-stranded cyclization reaction solution was added to the PCR tube after the reaction in step (8), vortexed and shaken for 6 times, 3 seconds each time, and the reaction solution was collected to the bottom of the tube by instantaneous centrifugation.

3) Placing the PCR tube of the step S3 on a PCR instrument, and performing the reaction according to the conditions in the following table 14:

TABLE 14

Temperature of	Time of day
		Hot lid	On
37℃	30min
		4℃	Hold

4) After the reaction is finished, taking out the PCR tube and immediately carrying out the next reaction.

(3) Digestion by enzyme

1) The digestion reaction was prepared on ice as shown in table 15 below:

watch 15

Components	Volume of
		Digestion Buffer	1.4μL
Digestion Enzyme	2.6μL
		Total	4μL

2) And (3) adding the 4 mu L enzyme digestion reaction solution into the PCR tube reacted in the step (10), vortexing and shaking for 6 times, 3s each time, and collecting the reaction solution to the bottom of the tube through instantaneous centrifugation.

3) Placing the PCR tube obtained in the step 2) on a PCR instrument, and carrying out reaction according to the conditions in the following table 16:

TABLE 16

Temperature of	Time of day
		Hot lid	On
37℃	30min
		4℃	Hold

4) After the reaction was complete, 7.5. mu. L digest Stop Buffer was added to each reaction, vortexed 6 times for 3s each, and the reaction solution was collected to the bottom of the tube by flash centrifugation and transferred to a new 1.5m L EP tube.

(4) Purification of the cyclization product

1) The DNA Clean Beads are taken out 30min in advance and placed at room temperature, and are fully shaken and uniformly mixed before use.

2) Pipette 170 μ L DNA Clean Beads into the product of step 3.3.4, gently pipette at least 10 times until fully mixed, the last time ensuring that all liquid and Beads in the tip are pipetted into a 1.5m L EP tube.

3) Incubate at room temperature for 10 min.

4) Transient centrifugation, 1.5m L EP tube placed in magnetic rack, standing for 2-5min until the liquid is clear, carefully pipetting and discarding the supernatant.

5) The 1.5m L EP tube was kept on the magnetic stand, the beads and tube walls were rinsed by adding 500. mu. L freshly prepared 80% ethanol, and the supernatant carefully aspirated and discarded.

6) And (5) repeating the step to suck the liquid in the tube as dry as possible.

7) Keeping the 1.5m L EP tube fixed on the magnetic frame, opening the tube cover of the 1.5m L EP tube, and drying at room temperature until the surface of the magnetic bead has no reflection and no crack.

8) The 1.5m L EP tube was removed from the magnetic stand, DNA was eluted by adding 22. mu. L TE Buffer and gently pipetting at least 10 times until well mixed.

9) Dissolve at room temperature for 10 min.

10) Transient centrifugation, placing 1.5m L EP tube on magnetic rack, standing for 2-5min until the liquid is clear, transferring 20 μ L supernatant to a new 1.5m L EP tube.

(5) Quality inspection of cyclization product

1) Use of

And (3) quantifying the cyclized and purified product according to the operation instruction of the quantitative Kit by using the ssDNA Assay Kit single-stranded DNA fluorescent quantitative Kit. The desired molar yield of the final cyclization product is > 80fmol and can be calculated with reference to Table 1 or according to equation 1.

Equation 1 conversion between the number of moles of a single-link chain and the mass

80fmol single chain loop corresponding to the mass (ng) 0.08 × DNA major fragment size (bp)/1000bp × 330ng

TABLE 17 corresponding yields for different PCR product fragment sizes 80fmol single-stranded loop

Fig. 1 shows a 2100 quality control diagram of high-throughput sequencing library of exosome RNA, fig. 2 shows a L abChip peak diagram of exosome RNA, and based on table 17 and the results of fig. 1 and 2, it can be seen that circular DNA constructed by the method provided in this example meets the sequencing requirements.

Experimental example 1

In this example, sequencing verification was performed on the high-throughput sequencing library of exosome RNA constructed in example 1, and the sequencing platform used was MGI and Gene + Seq sequencing platform. The results are shown in FIG. 3 and in Table 18 below:

TABLE 18 exosome library sequencing quality control results

The results of data quality control of 7 exosome libraries sequenced by Hiseq3000 platform or MGI2000 platform are shown in table 18. As can be seen from the table, there was no significant difference in GC content, QC30, alignment for the MGI2000 platform and Hiseq3000 platform, however, the percentage coverage of sequencing for the MGI2000 platform was significantly better than the HS3000 platform (p < 0.05). FIG. 3 shows the ratio of different RNA types in the sequencing of exosome RNA, and it can be seen from FIG. 3 that the high-throughput sequencing library of exosome RNA provided by the present invention has wider RNA coverage.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.

Sequence listing

<110> Beijing Jiyin technologies Ltd

Construction method, device and application of high-throughput sequencing library of <120> exosome RNA

<130>HA202000040

<160>4

<170>SIPOSequenceListing 1.0

<210>1

<211>30

<212>DNA

<213> Artificial sequence (Adaptor1)

<400>1

gaacgacatg gctacgatcc gacttgtggt 30

<210>2

<211>34

<212>DNA

<213> Artificial sequence (Adaptor2)

<400>2

ccacaagtcg gaggccaagc ggtcttagga agac 34

<210>3

<211>70

<212>DNA

<213> Artificial sequence (forward primer)

<400>3

tctcagtacg tcagcagttt agaggacaac aactccttgg ctcacagaac gacatggcta 60

cgatccgact 70

<210>4

<211>53

<212>DNA

<213> Artificial sequence (reverse primer)

<400>4

ggcatggcga ccttatcagt tgtcctctat tgtcttccta agaccgcttg gcc 53

Claims

1. A construction method of a high-throughput sequencing library of exosome RNA is characterized by comprising the following steps:

s1, extracting total RNA of the exosome;

s2, fragmenting the total RNA to obtain fragmented RNA;

s3, reverse transcribing the fragmented RNA into double-stranded cDNA;

2. The building method according to claim 1, wherein the step S3 includes:

s31, reverse transcribing the fragmented RNA into double-stranded cDNA;

s33, performing end repair on the purified double-stranded cDNA, and adding a base A to the 5 'end and the 3' end of the purified double-stranded cDNA to obtain the end-repaired double-stranded cDNA.

3. The building method according to claim 1 or2, wherein the step S4 includes:

4. The method of constructing according to any one of claims 1 to 3, wherein the total RNA comprises: mRNA, miRNA, lncRNA, snRNA, snoRNA.

5. The construction method according to any one of claims 1 to 4, wherein the sequencing linker is a Y-type linker formed by partially complementing a first sequence and a second sequence, the first sequence is a single-stranded nucleotide having a nucleotide sequence shown in SEQ ID No.1, and the second sequence is a single-stranded nucleotide having a nucleotide sequence shown in SEQ ID No. 2;

6. The constructing method according to any one of claims 1 to 5,

7. The method of constructing a double-stranded cDNA according to any one of claims 1 to 6, wherein the reaction solution for End-repairing the double-stranded cDNA comprises a reaction solution containing the double-stranded cDNA, 50. mu. L, NEB Next Ultra II End PrepReaction Buffer, 7. mu. L, NEB Next Ultra II End Prep Enzyme Mix, 3. mu. L;

the reaction solution for connecting the double-stranded cDNA with the 5 'joint and the 3' joint comprises a reaction solution containing double-stranded cDNA with the end repaired, 60 mu L, NEB Next L alignment Enhancer, 1 mu L, NEB Next Ultra II L alignment MasterMix, 30 mu L, 5 'joint and 3' joint, 1.5 mu M and 2 mu L;

8. A high throughput sequencing library of exosome RNAs, wherein the sequencing library is obtained based on the construction method of any one of claims 1 to 7.

9. An apparatus for constructing a high throughput sequencing library of exosome RNAs, comprising:

an extraction module for extracting total RNA of exosomes;

10. Any one of the following uses a1-a 6:

a1, use of the construction method of any one of claims 1-7 in exosome RNA sequencing;

a2, use of the method of construction of any one of claims 1-7 in the construction of an exosome RNA sequencing library;

a3, use of the high throughput sequencing library of claim 8 in exosome RNA sequencing;

a4, use of the device of claim 9 in exosome RNA sequencing;

a5, use of the device of claim 9 in the construction of an exosome RNA sequencing library;

a6, the method of construction as claimed in any one of claims 1 to 7, the high throughput sequencing library as claimed in claim 8 or the device as claimed in claim 9, for use in the preparation of products for early stage screening, prognostic assessment, and/or monitoring of clinical efficacy.