CN114958996B - Ultrahigh-throughput unicellular sequencing reagent combination - Google Patents
Ultrahigh-throughput unicellular sequencing reagent combination Download PDFInfo
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Abstract
The invention discloses an ultrahigh-flux single-cell sequencing reagent combination, which is used for carrying out intracellular reverse transcription on cells by using a molecular marker microbead, a reverse transcription sequence and a bridging primer, then enabling the cells subjected to the intracellular reverse transcription to be in a separated space with one or more cells by using a microplate technology or a microfluidic technology, cracking the cells under the action of a lysis solution, connecting the sequence obtained after the reverse transcription with the molecular marker sequence on the molecular marker microbead with the help of the bridging primer, obtaining a large number of sequences by PCR amplification, constructing and obtaining a cDNA sequencing library, and then carrying out high-flux sequencing to obtain specific transcriptome information of millions of single cells by one-time sequencing. Greatly improves the flux of single cell sequencing.
Description
Cross Reference to Related Applications
The application is a divisional application of Chinese patent application with the application number of 2021105177506, the application date of 2021, 05 and 12 months, entitled "an ultrahigh-flux single-cell sequencing method".
Technical Field
The invention relates to the technical field of single cell sequencing, in particular to an ultrahigh-flux single cell sequencing reagent combination.
Background
Since the Single Cell sequencing technology was proposed in Shang Fuchou, single Cell high throughput sequencing platforms were continuously introduced as Well as the wet shoots, such as the predominantly microfluidic Drop-Seq (Macosko, e.z., et al, high Parallel Genome-with Expression Profiling of industrial Cells Using nano-drivers 3562 (5): p.1202-1214) and inDrop-Seq (Klein, all m., et al, dropping for Single-Cell transcriptors Applied to bacterial Cells, cell 4324 (5): p.1187-1201) platforms, the predominantly microplate micro-Well-tissue (x.7, hand x.45, cell 3245, cell 3214, and Cell 3214. G. However, in the case of a microplate, when the amount of cells to be put into the microplate reaches a certain level, it is easy to cause two or more cells in one microwell, which results in contamination of cell-to-cell transcripts. To avoid this phenomenon, it was found by analysis that the capture of only one cell per bead was achieved when the ratio of the number of wells in the microplate was about 1/10 of the total number of wells in the microplate. However, this results in a large number of droplets or microwells with only empty microbeads and no cells, which greatly reduces the efficiency of the experiment and increases the cost of the experiment, resulting in that the cell flux of a single experiment of these platforms can be in units of ten thousand. However, in the case of mice and humans, the total cell amount of an individual species exceeds trillions, and when the platform throughput is far from the requirement of sequencing, a large amount of biological information is easily lost due to insufficient sequencing throughput, so that it is important to improve the throughput of single sequencing.
In recent years, ultra-high throughput techniques using individual cells as independent reaction systems have been developed, such as sci-RNA-seq ([ 1] Cao, J., et al., comparative Single-cell transcriptional profiling of a multicell organization. Science,2017.357 (6352): p.661-667.[2] Cao, J., et al., the Single-cell transcriptional marking of a mammalian organic labeling system, nature,2019.566 (7745): p.496-502) and SPLIT-seq (Rober, A.B., et al., single-cell transcriptional profiling of The depletion vascular access, scientific coding, and subsequent Single-cell transcriptional labeling using a Single-spot marker, which is labeled with a Single molecular marker, followed by a Single-cell transcriptional labeling loop 3763, scientific labelling, which is performed by using a Single-cell transcriptional labelling loop 3763, scientific labelling, 3785, and subsequent multiple-cell labeling. However, these methods are based on ligation reactions in cells, and therefore have problems such as low reaction efficiency, high contamination rate due to easy leakage of transcripts between cells, and the like, and thus the method has low practicability.
Paul et al (Datlinger, P., et al, ultra-high through high throughput single-cell RNA sequencing by combinatorial fluidic indexing. BioRxiv, 2019.) of the research center for molecular medicine of the Austrian academy of sciences, 12 months in 2019, carried a round of molecular markers containing 96/384 molecules to the fixed cells by reverse transcription, and then, the throughput of sequencing single cells was increased by 15 times by combining the microfluidic method. However, the sequencing platform cell based on microfluidics is not tested in parallel, the batch effect problem is obvious, and the defects of expensive equipment, difficult carrying, high sequencing cost and the like exist.
Disclosure of Invention
The invention provides an ultrahigh-throughput single-cell sequencing method, which can obtain specific transcriptome information of millions of single cells at one time.
An ultra-high throughput single cell sequencing method, comprising the steps of:
(1) The following reagents were prepared:
a) The molecular marker microbead comprises a microbead body and a coupled molecular marker sequence, wherein the molecular marker sequence comprises sequentially arranged components:
a universal primer sequence as a primer binding region during PCR amplification;
a first cell tag sequence;
a first bridging sequence;
b) A reverse transcription sequence for use in intracellular reverse transcription, the reverse transcription sequence comprising, in order:
a second bridging sequence;
the second cell tag sequence is matched with the first cell tag sequence to form a cell tag sequence, and the cell tag sequence is used for identifying cells from which mRNA corresponding to each sequence in the constructed sequencing library is taken;
the molecular tag sequence is used for identifying mRNA corresponding to each sequence in the constructed sequencing library;
a poly-T tail for complementary pairing with mRNA having a poly-A sequence in a cell;
c) A bridging primer for connecting the labeling sequence in the a) and the reverse transcription sequence in the b), wherein the two ends of the bridging primer are provided with sequences which are complementary and matched with the first bridging sequence and the second bridging sequence respectively;
(2) Adding a reverse transcription sequence into a cell sample to be sequenced to perform intracellular reverse transcription, and connecting a poly-T tail end of the reverse transcription sequence with a cDNA sequence obtained by reverse transcription of an intracellular mRNA sequence to obtain a reverse transcription sequence-cDNA sequence;
(3) Enabling the cells subjected to intracellular reverse transcription in the step (2) to be in a separated space with one or more cells through a microplate technology or a microfluidic technology, cracking the cells under the action of a lysis solution, incubating, respectively complementarily pairing the first bridging sequence and the second bridging sequence through a bridging primer, and then connecting the first bridging sequence and the second bridging sequence by using ligase to obtain a molecular marker sequence-reverse transcription sequence-cDNA sequence coupled with the microbeads;
(4) Collecting microbeads coupled with a molecular marker sequence, a reverse transcription sequence and a cDNA sequence, and carrying out PCR amplification to obtain a cDNA sequence with a first cell tag sequence, a second cell tag sequence and a molecular tag sequence;
(5) Constructing a cDNA sequencing library by using the product obtained in the step (4), and then carrying out high-throughput sequencing to obtain specific transcriptome information of millions of single cells.
By dividing the cell tag sequence into a first cell tag sequence and a second cell tag sequence, the second cell tag sequence is introduced in a reverse transcription sequence of each mRNA during reverse transcription in the cells, so that when one molecularly-labeled microbead is combined with a plurality of cells, the second cell tag sequence can be distinguished, otherwise, the sequences of a plurality of cells which are combined with the same molecularly-labeled microbead cannot be distinguished by only depending on the first cell tag sequence on the molecularly-labeled microbead. In the prior art, in order to avoid that one molecular marker microbead binds to multiple cells, strict control of conditions is required, for example, micropores in a microplate of an experiment are prepared to the size of accommodating only one molecular marker microbead and one cell as much as possible (in this case, the relative sizes of the molecular marker microbead and the cell are not too large, otherwise, it is not easy to realize that one molecular marker microbead binds to one cell), and meanwhile, the hole drop rate of the cell needs to be controlled at a low level so that the cell is sufficiently dispersed, but the situation that one molecular marker microbead binds to multiple cells cannot be avoided, and the sequence in this situation can be misjudged as coming from the same cell by a final sequencing result.
Meanwhile, the second cell tag sequence is matched with the first cell tag sequence to form a cell tag sequence, so that the combination quantity of the cell tag sequences is increased, and the detection of a larger number of cells can be realized at one time.
Preferably, the coupling mode of the microbeads and the molecular marker sequences is as follows: amino is used for replacing hydroxyl on the C6 position of the nucleotide at the 5' end of the molecular marker sequence, carboxyl is modified on the surface of the microbead, and the carboxyl and the amino are coupled through condensation. Because the molecular marker sequence is single-stranded oligonucleotide, the hydroxyl on the first nucleotide at the 5' end of the molecular marker sequence is replaced by amino, the carboxyl is modified on the surface of the microbead, and the molecular marker sequence is coupled to the microbead through the reaction of the amino and the carboxyl.
Preferably, the molecular tag sequence is at least partially a randomly synthesized random sequence.
More preferably, the first cell tag sequence comprises a plurality of specific fragments, the second cell tag sequence comprises at least one specific fragment, the specific fragments at different positions are selected from the same or different specific fragment libraries, and the first cell tag sequence and the second cell tag sequence identify cells differently by using the arrangement combination of the specific fragments.
More preferably, the preparation method of the molecular marker microbead comprises the following steps:
(1) The primer for synthesizing the molecular marker sequence is divided into a plurality of primers according to the number of the specific fragments, each primer comprises a specific fragment, and a joint sequence for bridging connection and complementation is arranged between the primers, wherein the primer corresponding to the 5 'end of the molecular marker sequence also comprises the universal primer sequence, and the primer corresponding to the 3' end of the molecular marker sequence also comprises the first bridging sequence;
(2) Coupling the primer corresponding to the 5' end of the molecular marker sequence with the microbead body, then sequentially annealing and extending the rest primers by a PCR method, and sequentially connecting the rest specific fragments of the molecular marker sequence in series from the 5' end to the 3' end to prepare the molecular marker microbead.
Preferably, the molecular marker sequence is: 5'-TTTAGGGATAACAGGGTAATAAGCAGTGGTATCAACGCAGAGTACGTNNNNNNCGACTCACTACAGGGNNNNNNTCGGTGACACGATCGNNNNNNTCGTCGGCAGCGTC-3', wherein N represents any one of A/T/C/G, and is randomly synthesized;
the reverse transcription sequence is as follows: 5' - [ phos)]ACACTCTTTCCCTACACGACGNNNNNNNNNNNNNNNNNNNNNTTTTTTTTTTTTTTTTVN-3 ', wherein the 5' end is added with a phosphorylation modification to provide a phosphate group for the ligation reaction; n represents any one of A/T/C/G and is randomly synthesized; n represents any one of A/T/C/G, and is randomly synthesized; v at the 3' end represents any one of A/C/G, and V is randomly synthesized;
the bridging primer is: 5' -CGTCGTGTAGGGAAAGAGTGTGACGCTGCCGACGA[ddC]-3', ddC is a dideoxycytidine modification.
In the reverse transcription sequence, a random sequence of 6 XN is used as a second cell tag sequence, and a random sequence of 10 XJ is used as a molecular tag sequence.
Preferably, the cell sample to be sequenced contains 2 or more than 2 cells. The ultrahigh-flux single cell sequencing method can realize simultaneous sequencing of various cells.
Preferably, the bead bodies are magnetic beads,
in the step (3), the cells subjected to intracellular reverse transcription in the step (2) are added into a microplate, and then the molecular marker microbeads are added, wherein the diameter of each micropore in the microplate is just large enough to accommodate one molecular marker microbead and one or more cells;
in the step (3), the hole dropping rate of the cells added into the microporous plate is controlled to be more than 80 percent; the hole falling rate of the molecular marker microbeads added into the microporous plate is more than 99 percent.
Due to the fact that the ultrahigh-flux single-cell sequencing method can achieve the effect that one molecular marker microbead is combined with a plurality of cells, when the microbead body is a magnetic bead and a microporous plate method is used, the falling hole rate can be greatly improved when the cells are added into the microporous plate.
The invention can be used for the single cell sequencing platform of a micro-fluidic control method besides the single cell sequencing method of a microplate method in which the bead bodies are magnetic beads.
More preferably, the depth of the micropores in the microplate is 30-160 μm, and the diameter of the micropores is 20-150 μm; the diameter of the bead body is 20-145 μm.
More preferably, the preparation method of the microplate comprises the following steps:
(1) Etching a micropore on a silicon wafer as an initial mold;
(2) Pouring polydimethylsiloxane on the initial mould, and taking down the polydimethylsiloxane after molding to form a secondary mould with microcolumns;
(3) Pouring hot-melt agarose with the mass volume ratio of 4-6% on a second mould, cooling and forming, and taking down the agarose.
The method comprises the steps of using a molecular marker microbead, a reverse transcription sequence and a bridging primer, carrying out intracellular reverse transcription on cells by using the reverse transcription sequence, enabling the cells subjected to intracellular reverse transcription to be in a separated space with one or more cells by a microplate technology or a microfluidic technology, cracking the cells under the action of a lysis solution, connecting a sequence obtained after reverse transcription with the molecular marker sequence on the molecular marker microbead with the help of the bridging primer, obtaining a large number of sequences through PCR amplification, constructing and obtaining a cDNA sequencing library, and carrying out high-throughput sequencing to obtain specific transcriptome information of millions of single cells through one-time sequencing. Greatly improves the flux of single cell sequencing.
Drawings
FIG. 1 is a schematic view of a honeycomb-arranged microplate.
FIG. 2 is a flow chart of the preparation of the molecular marker magnetic beads.
FIG. 3 is a schematic view of cells being dropped into a microplate.
FIG. 4 is a flow chart of cDNA library construction, wherein the universal sequences include the universal primer sequence, cell tag sequence 1, linker sequence 1, cell tag sequence 2, linker sequence 2, and cell tag sequence 3 as shown in FIG. 2.
FIG. 5 is a size distribution diagram of the prepared cDNA sequencing library fragments.
FIG. 6 is a comparison of human and mouse mixed cell populations.
FIG. 7 is a plot of sequencing reads/gene comparisons for different lysates.
FIG. 8 is a diagram showing the results of tSNE analysis of mouse testicular cells.
Detailed Description
Example 1
1. Microplate preparation
The microplate size (well plate size 1.8cm × 1.8 cm) was designed according to the experimental scale (50 ten thousand each of human 293T cells and mouse 3T3 cells), and micropores of a cylindrical shape having a depth of 60 μm, a diameter of 50 μm, and a hole pitch of 70 μm were etched on a silicon wafer as an initial mold. Then, polydimethylsiloxane (PDMS) was poured onto a silicon wafer, the PDMS was removed after molding to form a second mold having micro-pillars on the plate, the micro-pillars used in the final experiment were made of agarose (prepared with non-enzymatic water) with a concentration of 5% (mass ratio), the plate was melted and poured onto the PDMS micro-pillars plate to be condensed and molded, and the plate was removed to form a micro-pillar having a certain thickness (fig. 1). When in storage, the DPBS-EDTA mixed solution harmless to cells is added, and the mixture is stored in a refrigerator at 4 ℃ by covering, so that the good working state of the microporous plate can be ensured when the mixture is used.
2. Preparation of molecular marker magnetic beads
The magnetic beads were purchased from Suzhou knoyi microsphere science and technology Limited (product number MagCOOH-20190725) and surface carboxyl-coated, 45 μm in diameter. The preparation process of the molecular marker magnetic beads is shown in figure 2, and comprises 4 steps:
(1) Designing a molecular marker sequence, dividing the molecular marker sequence into three sections, arranging a joint sequence for connecting the two adjacent sections through PCR between the two adjacent sections, wherein the first section from 5' comprises a universal primer sequence and a partial cell tag sequence, the last section comprises a partial cell tag sequence, a whole molecular tag sequence and a bridging complementary sequence, and the rest sequences except the first section are complementary sequences of corresponding sequences.
(2) The sequence of each segment is as follows:
wherein 6 XN is the core sequence of the cell tag sequence, the core sequence corresponding to each magnetic bead is different, and the 6 XN sequences in three sequences corresponding to the same magnetic bead are also different, and because each site has 4 choices of A/T/C/G, the 6 XN sequence can have 4 6 And (4) selecting. N represents any of A/T/C/G, and is randomly synthesized.
(3) And respectively synthesizing all sequences, wherein 96 sequences are designed in all sequences belonging to the cell tag sequence part, each sequence is independently placed, and the hydroxyl group is replaced by the amino group at the C6 position of the nucleotide at the 5' end of the first segment of the sequence.
(4) Respectively coupling equivalent magnetic beads with 96 first-stage sequences, collecting 96 modified magnetic beads, uniformly mixing, uniformly dividing into 96 equal parts, mixing with 96 second-stage sequences, performing PCR sequence extension, uniformly dividing into 96 equal parts, mixing with 96 third-stage sequences, performing PCR sequence extension, and performing denaturation and melting to obtain 96 multiplied by 96 single-stranded oligonucleotide modified magnetic beads.
After completion, the sequence of the molecular marker is as follows: 5'-TTTAGGGATAACAGGGTAATAAGCAGTGGTATCAACGCAGAGTACGTNNNNNNCGACTCACTACAGGGNNNNNNTCGGTGACACGATCGNNNNNNTCGTCGGCAGCGTC-3'.
Example 2
Human 293T, murine 3T3 mixed cell assay.
5-10ml of methanol (precooled at 20 ℃) is slowly dripped into each 500 ten thousand of mouse Embryonic Stem Cells (ESC) 3T3 and human embryonic kidney cells (293T) respectively and fixed for 30 minutes at-20 ℃, meanwhile, the bridging primers are subpackaged into eight-connected tubes with 6.5 mu l of each hole and then are subpackaged into 96-well plates containing 0.5 mu l of reverse transcription primers, and the reverse transcription mixed primers with 1 mu l of each hole are formed by standing and mixing evenly. The bridging primer sequence is 5-CGTCGTGTAGGGAAAGAGTGTGACGCTGCCGACGA[ddC]The 3',3' end is modified by ddC to prevent the generation of by-products caused by extension during reverse transcription of the bridged primer.
The reverse transcription primer (reverse transcription sequence) and the cell label sequence are also 96, the core sequence is 6 XN, each primer is independently placed in each hole, the 6 XN random sequence can be used as a part of the cell label sequence, and the subsequent combination with the cell label sequence on the molecular marker magnetic beads in the embodiment 1 is used for identifying the cells from which the mRNA corresponding to each sequence in the subsequent constructed sequencing library is taken, so that 96X 96 single-stranded oligonucleotides are used for identifying the cells, and the application is enough for disposable million-level cells. The 5 'end of the molecular tag sequence of the last segment of the reverse transcription primer is added with phosphorylation modification to provide a phosphate group for a ligation reaction, N in 10 xn represents any one of A/T/C/G and is randomly synthesized, V at the 3' end represents any one of A/C/G, N represents any one of A/T/C/G, the random synthesis is mainly used for enabling the primer to be combined to the tail end of the polyA tail and avoiding being combined to the middle part of the polyA tail, and the specific sequence is as follows: 5' - [ phos)]ACACTCTTTCCCTACACGACGNNNNNNnnnnnnnnnnTTTTTTTTTTTTTTTTTTTTTTTTTVN-3’。
Configuration 310. Mu.l reverse transcriptionThe system (50. Mu.l dNTP, 200. Mu.l buffer, 50. Mu.l reverse transcriptase, 10. Mu.l RNase inhibitor) was mixed well and dispensed into 96-well plates containing reverse transcription mix primers, 3.1. Mu.l per well. The two cells after fixation were then washed by centrifugation once at 500g, and 250 ten thousand cells were mixed. The mixed cells (about 5 ten thousand per well) were aliquoted into 96-well plates containing a premixed reverse transcription system (6. Mu.l cell suspension, 0.5. Mu.l dNTP, 1. Mu.l reverse transcription mix primer, 2. Mu.l buffer, 0.5. Mu.l reverse transcriptase, 0.1. Mu.l RNase inhibitor) and reacted at 42 ℃ for 1.5 hours. After completion of the reverse transcription, the cells in the 96-well plate were collected by a discharge gun into an eight-tube, and then collectively transferred to a clean 1.5ml EP tube, washed once with a DPBS solution (Gibco, cat. No. 14190-144), and centrifuged at 500g for 5 minutes. After the supernatant was aspirated, the cells were resuspended in 500. Mu.L of DPBS solution and the cell suspension was added dropwise to the plate, resulting in more than 80% of the wells being filled with cells (FIG. 3). Adding 20 ten thousand molecular marker magnetic beads into the micro-porous plate with the cells, placing the micro-porous plate on a magnet, slightly mixing the micro-porous plate and the magnet to ensure that more than 99 percent of micro-pores are covered by the magnetic beads, and washing away redundant molecular marker magnetic beads by using a DPBS solution. 200. Mu.L of lysis buffer (ddH) was slowly added dropwise to the well-filled plate 2 O,10% SDS,50% formamide (vol/vol) and 3 XSSC), and incubated at room temperature for 30 minutes to allow the bridging sequence on the magnetic beads and the bridging primer on the cells to hybridize sufficiently complementarily. After completion of the incubation, the plate was inverted on a magnet, the magnetic beads with the molecular marker-mRNA complexes were collected, transferred to a 1.5ml EP tube, washed twice, the remaining liquid was aspirated with 20. Mu.l pipette gun, and 50. Mu.L of the ligation mixture (2. Mu.l of T4 ligase, 5. Mu.L of T4 buffer, 1. Mu.L of RNase inhibitor, 2. Mu.L of dNTP, 30% PEG80005. Mu.L, ddH) was added to the EP tube containing the molecular marker magnetic beads 2 O35 μ L), and left to react at 37 ℃ for 1 hour.
After the completion of the ligation reaction, the magnetic beads labeled with the molecules were washed three times on a magnetic rack, the supernatant was aspirated and reacted at 37 ℃ for 0.5 hour by adding 200. Mu.L of exonuclease EXONI mixed solution (EXONI buffer 1X, EXONI 1X), and the single-stranded nucleotide sequences on the magnetic beads which did not capture mRNA were removed. After the completion of the external cutting, the magnetic beads with the molecular markers are washed on a magnetic frame for three times, the supernatant is sucked and discarded, and 500 mu L of 0.1 percent NaOH solution is addedThe solution was treated for 5 minutes to obtain single-stranded cDNA for subsequent double-stranded synthesis reaction. The NaOH-treated molecularly-labeled magnetic beads were washed three times on a magnetic rack to remove residual NaOH, and then 100. Mu.L of the mixture for two-strand synthesis (20. Mu.L of reverse transcription buffer, 40. Mu.L 30% PEG8000, 10. Mu.L of 10mM dNTP, 10. Mu.L of 100. Mu.M random primer, 2.5. Mu.L of Klenow polymerase and 17.5. Mu.L of ddH were added 2 O) at 37 ℃ for 1 hour, wherein the random primer sequence is 5 '-AAGCAGTGGTATCACGCAGAGTGANNNGNNNB-3', B represents one of G/T/C, N represents any one of A/T/C/G, and the random synthesis. The random primer sequence will be bound randomly to the NaOH treated single stranded sequence of the magnetic beads and the polymerase will continue to synthesize the complementary strand along the 3' direction of the primer, where nnngnnb is the random binding sequence.
After the two-strand synthesis reaction was completed, the magnetic beads labeled with molecules were washed three times on a magnetic frame, the supernatant was aspirated off, and a PCR reagent mixture (KAPA HiFi Hot Start Ready Mix 1X, TSO-PCR primer 0.1. Mu.M) was added. Wherein, the TSO-PCR primer sequence is as follows: 5'-AAGCAGTGGTATCAACGCAGAGT-3'. PCR procedure: pre-denaturation at 98 ℃ for 3 min; denaturation at 98 ℃ for 20 seconds, annealing at 67 ℃ for 15 seconds, extension at 72 ℃ for 6 minutes, and repeating for 12 times; extending for 5 minutes at 72 ℃, and preserving heat at 4 ℃ to obtain a large amount of marked cDNA. PCR products were purified using norgestrel purification magnetic beads.
Before use, the purified magnetic beads are shaken and mixed evenly and are placed at room temperature for at least 30 minutes, and the purification steps are as follows:
(1) Adding 50 mu L of purified magnetic beads into the PCR reaction system, and uniformly mixing for more than 10 times by using a liquid transfer device to ensure that the whole system is uniform;
(2) Incubation for 10 minutes at room temperature;
(3) Placing the PCR tube on a magnetic frame for 5 minutes to ensure that the purified magnetic beads are completely adsorbed;
(4) Keeping the PCR tube on a magnetic frame, and carefully discarding the supernatant;
(5) Adding 200 mu L of freshly prepared 80% ethanol, incubating for 30 seconds, and then removing the supernatant;
(6) Repeating the steps once;
(7) Opening the cover, and drying in the air for 8 minutes;
(8) Adding 13. Mu.L of Elution buffer (Elution buffer) into the PCR tube to cover the purified magnetic beads, removing the PCR tube from the magnetic frame and resuspending the purified magnetic beads;
(9) Incubate for 2 minutes at room temperature and aspirate 12. Mu.L of final cDNA library (see FIG. 4 for the above reaction details);
(10) The size of the cDNA library fragments was analyzed by an Agilent 2100 Bioanalyzer (FIG. 5) and the resulting cDNA library fragments ranged from 300-1000bp.
A gene sequencing library was constructed by the method described in example 3 below, and the library was sent to Rapu for sequencing. And (3) splitting, screening and comparing data returned by sequencing to obtain a gene expression profile. The matrix file is imported into R language analysis, so that the matrix data can be converted into a visual graph. As can be seen from FIG. 6, there is very little contamination of double cells, and high throughput sequencing of single cells can be achieved.
Example 3
And constructing a cDNA sequencing library.
(1) 5ng initial DNA fragmentation
The kit TD512 from Vazyme was used.
(a) Thawing 5 XTTBL (TruePrep tag Buffer L) at room temperature, and reversing the upside down and mixing for later use. It was confirmed that 5 XTS (terminator Solution, reaction termination Solution) was at room temperature and whether or not precipitation was observed by flicking the tube wall. If the precipitate exists, the precipitate can be dissolved by heating at 37 ℃ and shaking vigorously and mixing the precipitate.
(b) Sequentially adding reaction components in a sterilized PCR tube:
(c) Gently flick 20 times by using a pipette and mix well.
(d) The PCR tube was placed in a PCR instrument and the following reaction program was set up: 10min at 55 ℃; keeping the temperature at 10 ℃.
(e) Immediately, 5. Mu.l of 5 XTS was added to the reaction product, and the mixture was gently pipetted and mixed well. Placing in a greenhouse for 5min.
(2) Enrichment by PCR
(a) Placing the sterilized PCR tube in an ice bath, and sequentially adding the reaction components:
the kit used is as follows: truePrep TM Index Kit V2 for
(Vazyme # TD 202), and the specific experimental procedures were according to the kit instructions.
(b) And (3) lightly blowing and beating by using a pipettor, fully and uniformly mixing, and placing the PCR tube in a PCR instrument for carrying out the following reaction: pre-denaturation at 72 ℃ for 3min and 98 ℃ for 30sec; deformation at 98 ℃ of 15sec, annealing at 60 ℃ for 30sec; extension for 3min at 72 ℃ for 11 cycles; storing at 4 ℃.
(3) Sorting and purifying the length of the amplified product
AMPure XP magnetic beads were used for length sorting and purification. The starting PCR product volume should be 50. Mu.l. Since the volume of the product was less than 50. Mu.l due to evaporation of the sample during PCR, the volume had to be made up to 50. Mu.l using sterile distilled water before proceeding with the following procedure, otherwise the sorting length would be inconsistent with the expectation. During the sorting process, the magnetic bead usage amounts (R1 and R2) in two rounds are as follows:
first round AMPure XP bead dose R1=30.0 μ l (0.60 ×) second round AMPure XP bead dose R2=7.5 μ l (0.15 ×)
Wherein the "x" numbers are all calculated based on the volume of the PCR product, such as "0.60 x" means 0.60 x 50 μ l =30.0 μ l.
(a) Vortex and shake the AMPure XP magnetic beads evenly and suck the volume of R1 into 50 mul of PCR product, and lightly blow and beat the product for 10 times by using a pipettor to mix the product evenly. Incubate for 5 minutes at room temperature.
(b) The reaction tube was centrifuged briefly and placed in a magnetic stand to separate AMPure XP beads and liquid. After the solution is clear (about 5 minutes), the supernatant is carefully transferred to a clean EP tube and the magnetic beads are discarded.
(c) Vortex, shake and mix AMPure XP magnetic bead and absorb R2 volume to supernatant, gently blow and beat 10 times using a pipettor and mix well. Incubate for 5 minutes at room temperature.
(d) The reaction tube was centrifuged briefly and placed in a magnetic stand to separate AMPure XP beads and liquid. The supernatant was carefully removed until the solution was clear (about 5 minutes).
(e) The EP tube was kept in the magnetic stand all the time, and 200. Mu.l of freshly prepared 80% ethanol was added to rinse the AMPure XP magnetic beads. After incubation at room temperature for 30 seconds the supernatant was carefully removed.
(f) Repeat step 5 for a total of two rinses.
(g) And keeping the EP tube in the magnetic frame all the time, and opening the cover to dry the AMPure XP magnetic beads for 10 minutes.
(h) The EP tube was removed from the magnetic stand and eluted with 13. Mu.l of sterile ultrapure water. Vortex or gently blow with a pipette to mix well. The reaction tube was centrifuged briefly and placed in a magnetic stand to separate AMPure XP beads and liquid. After the solution is clear (about 5 minutes), 12. Mu.l of the supernatant is carefully pipetted into a sterile EP tube to obtain a sequencing library, which is stored at-20 ℃.
Example 4
And (4) optimizing the cracking liquid with different ratios.
After the cell with the molecular tag was dropped onto the plate, as in example 2, the DPBS solution was used to wash off the excess of the molecularly labeled magnetic beads, and three different formulations of lysis solutions were added to three microwell plates, respectively, followed by control lysis solutions (0.1M Tris-HCl pH 7.5,0.5M LiCl,1% SDS,10mM EDTA and 5mM dithio, 1%) lysis solution 20 (ddH) 2 O,1% SDS,20% formamide and 3 XSSC), lysis solution 50 (ddH) 2 O,1% SDS,50% formamide and 3 XSSC), and incubated for 30 minutes by lysis, followed by the same procedures as in examples 2 and 3. Wherein SDS plays a main role in cleavage, formamide is used for promoting the hybridization of nucleic acid molecules, and SSC is used for assisting in improving the hybridization efficiency. The gene sequencing library was constructed and sent to rapp for sequencing, using the HiSeq2500PE125 sequencing strategy. The sequencing result is analyzed, and the lysate 50 remarkably improves the number of cells with UMI more than 500 and the average base factor (figure 7), and greatly improves the reaction efficiency of the platform.
Example 5
Analysis of mouse testis cells.
500 ten thousand of the digested mice were pooled according to the procedure of example 2, and a sequencing library was obtained according to example 3. The gene sequencing library was constructed and sent to rapp for sequencing, using the HiSeq2500PE125 sequencing strategy. And (3) splitting, screening and comparing data returned by sequencing to obtain a gene expression profile. The matrix file is imported into R language analysis, so that the matrix data can be converted into a visual graph. FIG. 8 shows the tSNE analysis of mouse testis cells, which shows that the mouse testis can be divided into 14 subgroups.
Example 6
The invention can also be applied to a single cell sequencing platform based on microfluidics. After obtaining fixed cells with one round of cell tags by the method described in example 2, the reaction was performed using 10X company chromium chip E (10X Genomics # 2000121). Inlet 1 injected with 75. Mu.l of fixed cells mixed with a ligation system (10. Mu.l cells, 50.5. Mu.l enzyme-free water, 7.5. Mu. l T4 ligation buffer, 3. Mu. l T4 ligase, 1.5. Mu.l 10 × Reducing Agent B and 2.5. Mu.l 100mM bridged primer), inlet 2 injected with 40. Mu.l of single-cell ATAC gel microbeads containing lysate (10Xgenomics #2000132, the microbead sequence as described in example 1), and inlet 3 injected with 240. Mu.l of Partioninating Oil (10Xgenomics # 220088). The emulsion-coated beads were then incubated at 37 ℃ for 1.5 hours to allow for complete ligation. After the reaction, the microbeads were collected and subjected to excision, single-stranded, double-stranded synthesis reactions according to the protocol described in example 2, and finally to PCR amplification and purification to obtain a cDNA library.
Sequence listing
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Claims (10)
1. An ultra-high throughput single cell sequencing reagent combination, which is characterized by comprising the following reagents:
a) The molecular marker microbead comprises a microbead body and a coupled molecular marker sequence, wherein the molecular marker sequence comprises sequentially arranged components:
a universal primer sequence as a primer binding region during PCR amplification;
a first cell tag sequence;
a first bridging sequence;
b) A reverse transcription sequence for use in intracellular reverse transcription, the reverse transcription sequence comprising, in order:
a second bridging sequence;
the second cell tag sequence is matched with the first cell tag sequence to form a cell tag sequence, and the cell tag sequence is used for identifying cells from which mRNA corresponding to each sequence in the constructed sequencing library is taken;
the molecular tag sequence is used for identifying mRNA corresponding to each sequence in the constructed sequencing library;
a poly-T tail for complementary pairing with mRNA having a poly-A sequence in a cell;
c) A bridging primer for linking the marker sequence in a) above to the reverse transcription sequence in b), the bridging primer having at both ends sequences complementary to the first and second bridging sequences, respectively,
the molecularly-labeled microbead, reverse transcription sequence and bridge primer are used for ultrahigh-throughput sequencing based on the following steps: enabling a molecular marker microbead and one or more cells to be in a separated space through a microplate technology after intracellular reverse transcription, cracking the cells under the action of a lysis solution, incubating, respectively complementarily pairing a first bridging sequence and a second bridging sequence through a bridging primer, and then connecting the first bridging sequence and the second bridging sequence by using a ligase to obtain a molecular marker sequence-reverse transcription sequence-cDNA sequence coupled with the microbead.
2. The ultra-high throughput single cell sequencing reagent combination of claim 1, wherein said microbead body and molecular marker sequence are coupled in a manner that: hydroxyl is substituted by amino on the C6 position of the nucleotide at the 5' end of the molecular marker sequence, carboxyl is modified on the surface of the microbead body, and the microbead body is coupled by condensation of the carboxyl and the amino.
3. The ultra-high throughput single cell sequencing reagent combination of claim 1, wherein said molecular tag sequence is at least partially a randomly synthesized random sequence.
4. The ultra-high throughput single cell sequencing reagent combination of claim 3, wherein said first cell tag sequence comprises specific fragments at a plurality of positions, said second cell tag sequence comprises specific fragments at least one position, the specific fragments at different positions are selected from the same or different libraries of specific fragments, and said first cell tag sequence and said second cell tag sequence identify cells differently using the permutation and combination of the specific fragments at different positions.
5. The ultra-high throughput single cell sequencing reagent combination of claim 4, wherein said method for preparing said molecularly-tagged microbeads comprises the steps of:
(1) The primer for synthesizing the molecular marker sequence is divided into a plurality of primers according to the number of the specific fragments, each primer comprises a specific fragment, and a joint sequence for bridging connection and complementation is arranged between the primers, wherein the primer corresponding to the 5 'end of the molecular marker sequence also comprises the universal primer sequence, and the primer corresponding to the 3' end of the molecular marker sequence also comprises the first bridging sequence;
(2) Coupling the primer corresponding to the 5' end of the molecular marker sequence with the microbead body, then sequentially annealing and extending the rest primers by a PCR method, and sequentially connecting the rest specific fragments of the molecular marker sequence in series from the 5' end to the 3' end to prepare the molecular marker microbead.
6. The ultra-high throughput single cell sequencing reagent combination of claim 1, wherein said molecular marker sequence is: 5'-TTTAGGGATAACAGGGTAATAAGCAGTGGTATCAACGCAGAGTACGTNNNNNNCGACTCACTACAGGGNNNNNNTCGGTGACACGATCGNNNNNNTCGTCGGCAGCGTC-3', wherein N represents any one of A/T/C/G, and is randomly synthesized;
the reverse transcription sequence is as follows: 5' - [ phos)]ACACTCTTTCCCTACACGACGNNNNnnnnnnnnnnTTTTTTTTTTTTTTTVN-3 ', wherein the 5' end is added with phosphorylation modification to provide a phosphate group for the ligation reaction;n represents any one of A/T/C/G and is randomly synthesized; n represents any one of A/T/C/G, and is randomly synthesized; v at the 3' end represents any one of A/C/G, and V is randomly synthesized;
the bridging primer is: 5' -CGTCGTGTAGGGAAAGAGTGTGACGCTGCCGACGA[ddC]-3', ddC is a dideoxycytidine modification.
7. The ultra-high throughput single cell sequencing reagent combination of claim 1, wherein said bead bodies are magnetic beads.
8. The ultra-high throughput single cell sequencing reagent combination of claim 7, further comprising a microplate and a lysis solution, wherein the depth of the microwell in the microplate is 30-160 μm, and the diameter of the microwell is 20-150 μm; the diameter of the bead body is 20-145 μm.
9. The ultra-high throughput single cell sequencing reagent combination of claim 8, wherein said microplate is prepared by a method comprising:
(1) Etching a micropore on a silicon wafer as an initial mold;
(2) Pouring polydimethylsiloxane on the initial mould, and taking down the polydimethylsiloxane after molding to form a secondary mould with microcolumns;
(3) Pouring hot-melt agarose with the mass volume ratio of 4-6% on a second mould, cooling and forming, and taking down the agarose to obtain the microporous plate.
10. The method of using the ultra-high throughput single-cell sequencing reagent combination of claim 1, comprising the steps of:
(1) Adding a reverse transcription sequence into a cell sample to be sequenced to perform intracellular reverse transcription, and connecting a poly-T tail end of the reverse transcription sequence with a cDNA sequence obtained by reverse transcription of an intracellular mRNA sequence to obtain a reverse transcription sequence-cDNA sequence;
(2) Enabling the cells subjected to intracellular reverse transcription in the step (1) to be in a separated space with one or more cells through a microplate technology, cracking the cells under the action of a lysis solution, incubating, respectively complementarily pairing the first bridging sequence and the second bridging sequence through a bridging primer, and then connecting the first bridging sequence and the second bridging sequence by using ligase to obtain a molecular marker sequence-reverse transcription sequence-cDNA sequence coupled with the microbeads;
(3) Collecting microbeads coupled with a molecular marker sequence, a reverse transcription sequence and a cDNA sequence, and carrying out PCR amplification to obtain a cDNA sequence with a first cell tag sequence, a second cell tag sequence and a molecular tag sequence;
(4) Constructing a cDNA sequencing library by using the product obtained in the step (3), and then carrying out high-throughput sequencing to obtain specific transcriptome information of millions of single cells.
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