阅读说明：本技术 一种真核生物低起始量转录组文库构建方法 (Method for constructing low-initial-quantity transcriptome library of eukaryote ) 是由 杨新鑫 刘艳艳 朱月艳 孙子奎 于 2020-06-10 设计创作，主要内容包括：本发明公开了一种真核生物低起始量转录组文库构建方法,其特征在于,包括了全长cDNA扩增步骤和转座酶建库步骤。将全长cDNA的扩增后的扩增产物在转座酶的作用下进行转录和建库。将才能达到低起始量建库成功的效果。将样本起始量在5-50ng或50ng以上的样本均能获得更好的建库结果,方便后续的样本检测。(The invention discloses a construction method of a low initial transcriptome library of a eukaryote, which is characterized by comprising a full-length cDNA amplification step and a transposase library construction step. And (3) transcribing and constructing a library of amplified products of the full-length cDNA under the action of transposase. The effect of successful library building with low initial quantity can be achieved. The sample with the initial amount of 5-50ng or more than 50ng can obtain better library building result, and the subsequent sample detection is convenient.)

1. A method for constructing a low initial transcriptome library of a eukaryote is characterized by comprising the following steps:

full-length cDNA amplification step:

firstly, carrying out hydrolysis denaturation on RNA of an acquired sample, and then carrying out reverse transcription to obtain a reverse transcription product;

incubating the reverse transcription product, performing cDNA amplification to obtain a cDNA amplification product, incubating and purifying, and performing quality control to obtain a final purified cDNA amplification product,

wherein the content of the first and second substances,

the reverse transcription reaction system comprises:

reverse transcriptase III and/or RNA hydrolase inhibitor and/or reverse transcriptase III first chain and/or reducing agent DTT and/or glycine betaine and/or MgCl2, reverse transcription primer, the reverse transcription primer is AAGCAGTGGTATCACGCAGAGTCATRACRGG + G;

the quality control comprises the following steps:

1) taking 1 mul of purified DNA product qubit for quantification;

2) taking a proper amount of sample to be diluted to 1 ng/mu l, and carrying out fragment detection;

transposase library building step:

according to the qubit quantitative result after cDNA amplification, selecting the final product of the full-length cDNA amplification step to carry out fragmentation operation of 50ng of the initial DNA purification product or 5ng of the initial DNA purification product, then carrying out PCR enrichment operation to obtain the PCR purification product, and then carrying out library quality detection to obtain the final product.

2. The method of claim 1, wherein the cDNA amplification is performed by adding a cDNA amplification system to the reverse transcription product, wherein the cDNA amplification system comprises:

KAPA HiFi high fidelity hot start DNA polymerase premix solution and \ or A-IS PCR primers and \ or nucleic-free water without Nuclease,

wherein, the amplification primer of the A-IS PCR primers IS AGCAGTGGTATCAACGCAGAGT.

3. The method for constructing the eukaryotic low-initial transcriptome library according to claim 1, wherein the step of detecting the quality of the library comprises:

the prepared library is subjected to concentration quantification by using the Qubit, Labchip GX Touch HT chip and DNAhigh Sensitivity kit are used for Labchip detection,

and if the quality inspection result shows that: the size of the main peak of the library peak pattern is between 300-550bp, the base line is smooth, the peak pattern is single and smooth, no hybrid peak, no large fragment, no joint and no primer dimer are generated, and the library quality and concentration are qualified.

4. The method for constructing a low initial transcriptome library of eukaryote according to claim 1, wherein said final product of the full-length cDNA amplification step is selected to be subjected to a fragmentation operation of 50ng of the initial DNA purification product or a fragmentation operation of 5ng of the initial DNA purification product, specifically a fragmentation operation of 50ng of the initial DNA purification product when the total amount of the cDNA full-length amplification product is not less than 50 ng;

and the total amount of cDNA full-length amplification product is between 5 and 50ng, and a fragmentation operation step of 5ng of the initial DNA purification product is adopted.

5. The method of claim 4, wherein the step of fragmenting 50ng of the initial DNA purified product comprises:

1) thawing 5 XTTBL at room temperature, reversing the top and the bottom, mixing the mixture evenly, and configuring a reaction system as shown in the following table I in a PCR tube:

watch 1

2) Gently pipetting 20 times by using a pipettor, fully mixing, placing in a PCR instrument, and running the reaction program of the following table two:

watch two

Temperature of Time of day 105℃ Hot lid 55℃ 10min 10℃ Hold

3) Fragmentation product usePurifying the DNA Clean Beads:

step 1, vortex oscillation and uniform mixing

step 2, centrifuging the reaction tube for a short time, placing the reaction tube on a magnetic rack, separating magnetic beads from liquid, and carefully removing supernatant after the solution is clarified;

step 3, keeping the reaction tube on a magnetic frame all the time, adding 200 mul of freshly prepared 80% ethanol to rinse the magnetic beads, incubating at room temperature for 30s, and carefully removing the supernatant;

step 4, repeating the step 3, and rinsing twice in total;

step 5, keeping the reaction tube on a magnetic frame all the time, and opening a cover to dry the air for about 5 min;

step 6, taking the reaction tube out of the magnetic frame, and adding 26 mul of sterilized ultrapure water for elution; vortexing or beating with a pipette for 10 times, mixing, and incubating at room temperature for 5 min;

and 7, centrifuging the reaction tube for a short time, placing the reaction tube on a magnetic rack, separating magnetic beads from liquid, carefully sucking 24 mu l of supernatant into a new sterilized PCR tube after the solution is clarified, and then carrying out PCR enrichment.

6. The method of claim 4, wherein the step of fragmenting 5ng of the initial DNA purified product comprises:

1) thawing 5 XTBL at room temperature, reversing the top and the bottom, mixing uniformly for later use, and configuring the following three reaction systems in a PCR tube:

watch III

2) Gently pipetting 20 times by using a pipettor, fully mixing, placing in a PCR instrument, and running the following four reaction programs:

watch four

Temperature of Time of day 105℃ Hot lid 55℃ 10min 10℃ Hold

3) Immediately after the reaction, 5. mu.l of 5 XTS was added to the product, and the mixture was gently pipetted and mixed well, and left at room temperature for 5min for PCR enrichment.

Technical Field

The invention relates to the technical field of molecular biology and biology, in particular to a method for constructing a low-initial-quantity transcriptome library of eukaryotes.

Background

The sequencing of the common eukaryotic transcriptome in the current market has higher requirement on the amount of RNA of a sample, so that a plurality of tissue samples produced by RNA in a small amount are difficult to meet the sequencing requirement, and the sequencing of the common transcriptome cannot be carried out. Such as: clinical samples such as stem cells and tumors with rare sources, rare archaeological samples and special samples with difficult RNA extraction and easy degradation.

Even if the common transcriptome library is built by the small amount of RNA produced by the RNA, the library building power, the library quality and the sequencing data of the tissue sample are greatly influenced, so that the insufficient amount of the RNA sample becomes one of the important bottlenecks for restricting the construction of the common eukaryote transcriptome library.

Disclosure of Invention

In order to overcome the above-mentioned defects of the prior art, the present invention aims to provide a method for constructing a low-initial-amount transcriptome library of eukaryotes.

In order to realize the purpose of the invention, the adopted technical scheme is as follows:

a method for constructing a low-initial-amount transcriptome library of eukaryote comprises the following steps:

full-length cDNA amplification step:

firstly, carrying out hydrolysis denaturation on RNA of an acquired sample, and then carrying out reverse transcription to obtain a reverse transcription product;

incubating the reverse transcription product, performing cDNA amplification to obtain a cDNA amplification product, incubating and purifying, and performing quality control to obtain a final purified cDNA amplification product,

wherein the content of the first and second substances,

the reverse transcription reaction system comprises:

reverse transcriptase III and/or RNA hydrolase inhibitor and/or reverse transcriptase III first chain and/or reducing agent DTT and/or glycine betaine and/or MgCl2, reverse transcription primer, the reverse transcription primer is AAGCAGTGGTATCACGCAGAGTCATRACRGG + G;

the quality control comprises the following steps:

1) taking 1 mul of purified DNA product qubit for quantification;

2) taking a proper amount of sample to be diluted to 1 ng/mu l, and carrying out fragment detection;

transposase library building step:

according to the qubit quantitative result after cDNA amplification, selecting the final product of the full-length cDNA amplification step to carry out fragmentation operation of 50ng of the initial DNA purification product or 5ng of the initial DNA purification product, then carrying out PCR enrichment operation to obtain the PCR purification product, and then carrying out library quality detection to obtain the final product.

In a preferred embodiment of the present invention, the cDNA amplification is adding a cDNA amplification system to the reverse transcription product, wherein the cDNA amplification system comprises:

KAPA HiFi high fidelity hot start DNA polymerase premix solution and \ or A-IS PCR primers and \ or nucleic-free water without Nuclease,

wherein, the amplification primer of the A-IS PCR primers IS AGCAGTGGTATCAACGCAGAGT.

In a preferred embodiment of the present invention, the library quality detection step is specifically:

the prepared library is subjected to concentration quantification by using the Qubit, Labchip GX Touch HT chip and DNA High Sensitivity kit are used for Labchip detection,

and if the quality inspection result shows that: the size of the main peak of the library peak pattern is between 300-550bp, the base line is smooth, the peak pattern is single and smooth, no hybrid peak, no large fragment, no joint and no primer dimer are generated, and the library quality and concentration are qualified.

In a preferred embodiment of the present invention, the final product of the full-length cDNA amplification step is alternatively subjected to a fragmentation operation of 50ng of the starting DNA purification product or a fragmentation operation of 5ng of the starting DNA purification product, specifically a fragmentation operation of 50ng of the starting DNA purification product when the total amount of the cDNA full-length amplification product is greater than or equal to 50 ng;

and the total amount of cDNA full-length amplification product is between 5 and 50ng, and a fragmentation operation step of 5ng of the initial DNA purification product is adopted.

In a preferred embodiment of the present invention, the fragmentation procedure of 50ng of the starting DNA purified product specifically comprises:

1) thawing 5 XTTBL at room temperature, reversing the top and the bottom, mixing the mixture evenly, and configuring a reaction system as shown in the following table I in a PCR tube:

watch 1

2) Gently pipetting 20 times by using a pipettor, fully mixing, placing in a PCR instrument, and running the reaction program of the following table two:

watch two

Temperature of	Time of day
		105℃	Hot lid
55℃	10min
		10℃	Hold

3) Fragmentation product usePurifying the DNA Clean Beads:

step 1, vortex oscillation and uniform mixingDNA Clean Beads are sucked into 50 mu l to 50 mu l of fragmentation products, are fully and uniformly mixed by vortex oscillation or blow beating for 10 times by using a pipettor, and are incubated for 5min at room temperature;

step 2, centrifuging the reaction tube for a short time, placing the reaction tube on a magnetic rack, separating magnetic beads from liquid, and carefully removing supernatant after the solution is clarified;

step 3, keeping the reaction tube on a magnetic frame all the time, adding 200 mul of freshly prepared 80% ethanol to rinse the magnetic beads, incubating at room temperature for 30s, and carefully removing the supernatant;

step 4, repeating the step 3, and rinsing twice in total;

step 5, keeping the reaction tube on a magnetic frame all the time, and opening a cover to dry the air for about 5 min;

and 6, taking the reaction tube out of the magnetic frame, and adding 26 mu l of sterilized ultrapure water for elution. Vortexing or beating with a pipette for 10 times, mixing, and incubating at room temperature for 5 min;

and 7, centrifuging the reaction tube for a short time, placing the reaction tube on a magnetic rack, separating magnetic beads from liquid, carefully sucking 24 mu l of supernatant into a new sterilized PCR tube after the solution is clarified, and then carrying out PCR enrichment.

In a preferred embodiment of the present invention, the fragmentation operation of 5ng of the starting DNA purified product specifically comprises:

1) thawing 5 XTBL at room temperature, reversing the top and the bottom, mixing uniformly for later use, and configuring the following three reaction systems in a PCR tube:

watch III

2) Gently pipetting 20 times by using a pipettor, fully mixing, placing in a PCR instrument, and running the following four reaction programs:

watch four

Temperature of	Time of day
		105℃	Hot lid
55℃	10min
		10℃	Hold

3) Immediately after the reaction, 5. mu.l of 5 XTS was added to the product, and the mixture was gently pipetted and mixed well, and left at room temperature for 5min for PCR enrichment.

The invention has the beneficial effects that:

the invention transcribes and constructs a library by the amplified product of the full-length cDNA under the action of transposase. The effect of successful library building with low initial quantity can be achieved. The sample with the initial amount of 5-50ng or more than 50ng can obtain better library building result, and the subsequent sample detection is convenient.

Drawings

FIG. 1 is a library length distribution test chart.

Detailed Description

The main principle of the invention is as follows:

the method breaks through the bottlenecks of high risk and low success rate of common transcriptome library construction of low initial amount or precious samples, realizes the efficient and stable transcriptome sequencing of the eukaryotic organisms with low initial amount (trace), and is favorable for promoting functional genomics research better.

Compared with the traditional common eukaryotic organism library construction method, the method has the following innovation and advantages:

1. the initial amount of total RNA of a required sample can be as low as 5-50ng, and the sample has no species preference, and is widely applied to eukaryotes such as animals, plants, fungi and the like;

2. taking the total RNA of a sample to be detected as a template, carrying out reverse transcription on mRNA in the total RNA into double-stranded cDNA of a full-length fragment by utilizing Superscript III reverse transcriptase, and amplifying the double-stranded cDNA to reach the amount of DNA required by library construction so as to ensure the smooth construction of a subsequent library;

3. the DNA fragmentation is carried out by adopting a novel transposase method, so that the library building step is simplified, the input amount of a cDNA template is obviously reduced, and the library building time is shortened.

Through the improvement and innovation, the method can enrich the complete full-length transcript with high sensitivity and no preference and amplify cDNA, maintain the abundance of each transcript of the original mRNA, simplify the fussy steps of DNA fragmentation, terminal repair, linker ligation reaction and the like into one-step simple enzymatic reaction, effectively reduce the workload in the library construction process, ensure the stability and the repeatability of the library construction to the maximum extent, greatly reduce the library construction cost and realize the construction of the eukaryotic organism micro sample transcriptome library.

The process of the invention is further illustrated below with reference to specific examples:

first, full-Length cDNA amplification

Denaturation of RNA

1) The following reaction systems were arranged in 0.2ml PCR tubes (run on ice) as shown in Table 1:

TABLE 1

Components	Addition amount (μ l)	Concentration of
			Total RNA (50ng)	2.51
3′CDS primer(10μM)	1	1μM
			dNTP(10mM)	1	1mM
RNA hydrolase inhibitor (40U/. mu.l)	0.05	2U
			Total content of	4.56

2) The sample is mixed evenly and placed in a PCR instrument, incubated for 3min at 72 ℃ (hot cover 105 ℃), and immediately stored on ice after the incubation is finished.

2. Reverse transcription

1) The reverse transcription reaction system was configured as follows in table 2:

TABLE 2

Wherein the involved reverse transcription primer is AAGCAGTGGTATCAACGCAGAGTACATrGrG + G; at the 3' end there are two ribo-guanosines (rG) and one LNA modified guanosine (+ G).

(i.e., the reverse transcription primer: AAGCAGTGGTATCAACGCAGAGTACATGGG.)

2) Adding 5.44 mul of reverse transcription reaction system into the solution after RNA denaturation reaction and incubation, blowing, uniformly mixing, placing in a PCR instrument after short-time centrifugation, and incubating according to the following reaction conditions in the table 3:

TABLE 3

3) After the reaction was completed, the reaction mixture was centrifuged for a short time and stored on ice.

Amplification of cDNA

1) The PCR amplification reaction was prepared on ice as follows in table 4:

the kit KAPA HiFi HotStartStreacyMix manufactured by Kapa biosystems brand, cat # KM2605 is used in this step

TABLE 4

Components	Adding	Concentration of
			KAPA HiFi hot start DNA polymerase premix	12.5	1×
A-IS PCR primers (10. mu.M)	1	0.1μM
			Nuclease-free water	1.5
Total content of	15

The amplification primer of the A-IS PCR primers IS AGCAGTGGTATCAACGCAGAGT; an Amine (NH2) is added at the C6 position at the 5' end.

2) Adding the prepared PCR amplification reaction system into the reaction solution after reverse transcription, blowing and uniformly mixing the reaction system of 25 mu l, putting the mixture into a PCR instrument, and incubating according to the parameters of the following table 5:

TABLE 5

4. Magnetic bead purification

1) After the amplification is finished, adding 20 ul (0.8X) of uniformly mixed magnetic beads into the PCR amplification product, gently sucking for 10 times, fully mixing uniformly, and incubating at room temperature for 5 min;

2) placing the PCR tube on a magnetic frame, incubating at room temperature for 5min, and discarding the supernatant;

3) adding 200 mul of 80% ethanol, keeping the PCR tube on a magnetic frame without falling off, incubating at room temperature for 30s, and discarding the waste liquid;

4) repeating the step 3) once;

5) drying at room temperature for 3-5min, and removing the PCR tube from the magnetic frame after observing that the surfaces of the magnetic beads are dry or not reflective;

6) adding 27.5 μ l of sterilized water, gently blowing and beating for 10 times, mixing, and incubating at room temperature for 2 min;

7) the PCR tube was placed on a magnetic frame, incubated at room temperature for 5min until the solution became clear, and 25. mu.l of the supernatant was transferred to a new PCR tube.

Quality control of cDNA

1) Taking 1 mul of purified DNA product qubit for quantification;

2) an appropriate amount of sample was diluted to 1 ng/. mu.l and subjected to fragment detection (Labchip or 2100).

Secondly, transposase library construction

The Kit TruePrep DNA Library Prep Kit V2for Illumina, 1-A adopted in the step and produced by Vazyme brand uses a product number TD 501; 1-B use order number: the TD 502.

1-A.50ng initial DNA fragmentation operation:

(A and B are selected alternatively, the selection standard is determined according to the qubit concentration determined in the previous step, in particular when the total amount of the cDNA full-length amplification products is more than or equal to 50ng, the step of selecting A is carried out, and the step of selecting B is carried out when the total amount of the cDNA full-length amplification products is between 5 and 50 ng.)

1) Thawing 5 × TTBL at room temperature, mixing the mixture by turning the mixture upside down, and placing a reaction system as shown in the following table 6 in a PCR tube:

TABLE 6

2) Gently pipetting 20 times by using a pipette, fully mixing, placing in a PCR instrument, and running the reaction program shown in the following table 7:

TABLE 7

Temperature of	Time of day
		105℃	Hot lid
55℃	10min
		10℃	Hold

Product purification should be done immediately after the reaction is complete, otherwise the DNA sample will be excessively fragmented, resulting in smaller final library fragments.

3) Fragmentation product usePurifying the DNA Clean Beads:

step 1, vortex oscillation and uniform mixingDNA Clean Beads were pipetted into 50. mu.l to 50. mu.l of the fragmented product, mixed well by vortexing or pipetting 10 times, and incubated at room temperature for 5 min.

Step 2, the reaction tube was centrifuged briefly and placed on a magnetic stand to separate the magnetic beads from the liquid, and the supernatant was carefully removed after the solution was clarified (5 min).

And 3, keeping the reaction tube on a magnetic frame all the time, adding 200 mu l of freshly prepared 80% ethanol to rinse the magnetic beads, incubating at room temperature for 30s, and carefully removing the supernatant.

And 4, repeating the step 3 and rinsing twice in total.

And 5, keeping the reaction tube on the magnetic frame all the time, and opening the cover to dry in air for about 5 min.

And 6, taking the reaction tube out of the magnetic frame, and adding 26 mu l of sterilized ultrapure water for elution. Vortexing or blowing and beating for 10 times by using a pipette, and fully mixing, and incubating for 5min at room temperature.

And 7, centrifuging the reaction tube for a short time, placing the reaction tube on a magnetic rack, separating magnetic beads from liquid, carefully sucking 24 mu l of supernatant into a new sterilized PCR tube after the solution is clarified (5min), and immediately carrying out the PCR enrichment in the step 2.

B.5ng initial DNA fragmentation operation:

1) thawing 5 XTTBL at room temperature, reversing the top and the bottom, mixing the mixture evenly, and configuring the following reaction system in a PCR tube as shown in the following table 8:

TABLE 8

2) Gently pipetting 20 times by using a pipette, fully mixing, placing in a PCR instrument, and operating the following reaction program of Table 9:

TABLE 9

Temperature of	Time of day
		105℃	Hot lid
55℃	10min
		10℃	Hold

3) Immediately after the reaction, 5. mu.l of 5 XTS was added to the product, and the mixture was gently pipetted and mixed well and left at room temperature for 5 min.

5 × TS should be added immediately after the reaction is complete, otherwise the DNA sample will be excessively fragmented, resulting in smaller final library fragments.

4) Step 2.PCR enrichment was performed immediately.

Enrichment by PCR

1) The PCR tube was placed on ice and the following reaction system was set up as in Table 10 below:

watch 10

Components	Volume of
		Fragmentation product + ddH2O	24μl
5×TAB	10μl
		PPM	5μl
N5XX*	5μl
		N7XX*	5μl
TAE	1μl

2) Using a pipette to gently blow and beat the mixture to be well mixed, placing the reaction tube in a PCR instrument, and operating the following reaction program in the following table 11:

TABLE 11

The number of amplification cycles is selected according to the actual situation, and the selection principle is as follows in table 12:

TABLE 12

Initial cDNA amount	Number of amplification cycles
		50ng	5--9
5ng	9--15

Purification of PCR products

1) Vortex oscillation mixing

DNA Clean Beads and pipette 50. mu.l (1X) into 50. mu.l PCR product, vortex or blow-beat 10 times using a pipette, mix well, incubate 5min at room temperature;

2) placing the PCR tube on a magnetic frame, and incubating for 5min at room temperature;

3) discarding the supernatant, and keeping the PCR tube on the magnetic rack without falling off, wherein the supernatant cannot touch the magnetic beads;

4) adding 200 μ l of 80% ethanol, incubating at room temperature for 30s, and discarding the waste liquid;

repeating the step 4) once.

5) Air-drying at room temperature for 3-5min, and removing the PCR tube from the magnetic frame when the surface of the beads is dry or not reflected;

6) adding 52.5 μ l of enzyme-free water, gently blowing and beating for 10 times, mixing, covering the PCR tube cover, and incubating at room temperature for 2 min;

7) placing the PCR tube on a magnetic frame, and incubating for 5min at room temperature;

8) transfer 50. mu.l of supernatant to a new PCR tube.

4. Library fragment sorting

1) Add 27.5. mu.l (0.55X) of magnetic beads to the 50. mu.l of purified DNA, blow and mix well, and leave at room temperature for 5 min;

2) placing the PCR tube on a magnetic frame, and standing for 5 min;

3) transfer 75ul of the supernatant to a new PCR tube, add 7.5. mu.l (0.15X) of magnetic beads, and stand at room temperature for 5 min;

4) placing the PCR tube on a magnetic frame, and standing for 5 min;

5) discarding the supernatant, adding 200 μ l of 80% ethanol, incubating at room temperature for 30s, and discarding the waste liquid;

6) repeating the step 5) once, and drying at room temperature for 3-5 min;

7) adding 27.5 μ l of enzyme-free water, blowing and mixing with a gun, and standing at room temperature for 2 min;

8) transfer 25. mu.l of eluted library product to a new tube and store at-20 ℃.

Third, quality detection of library

Taking 1ul of the prepared library, and using the Qubit to carry out concentration quantification; taking 1ul of the prepared library, using a PELabchip GX Touch HT chip and a DNA High Sensitivity kit to carry out labchip detection, and quality inspection results show that the size of the main peak of a library peak pattern graph is between 300 and 550bp, the base line is smooth, the peak pattern is single and smooth, no impurity peak, no large fragment, no joint and no primer dimer, and the quality and the concentration of the library are qualified.

1. Library concentrations are shown in table 13:

watch 13

2. Library length distribution assay figure 1, wherein the green line represents: an about 450bp library obtained by magnetic bead sorting; orange thread: an about 470bp library obtained by magnetic bead sorting;

purple line indicates: an about 500bp library obtained by magnetic bead sorting; blue line: the resulting about 520bp library was sorted by magnetic bead.

The invention is achieved by: on one hand, the amplification of full-length cDNA and on the other hand, the transposase is introduced into a transcriptome database, and the successful database construction with low initial amount can be achieved under the two aspects.

Sequence listing

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