CN113604545B - Ultrahigh-throughput single-cell chromatin transposase accessibility sequencing method - Google Patents

Abstract

The invention discloses an ultra-high flux single cell chromatin transposase accessibility sequencing method, firstly using specific molecular label transposase embedding compound to transpose chromatin transposase accessibility genome sequence in cell nucleus, then making a molecular mark microbead and one or more cell nuclei in separated space by using microplate technique or microfluidic technique, and cracking the cell nucleus under the action of lysis solution, connecting the specific molecular label sequence with the molecular marker sequence on the molecular marker microbead with the help of bridging primers, obtaining a large number of sequences through PCR amplification, constructing and obtaining a sequencing library, then high-throughput sequencing is carried out, the specific genome openness information of millions of single cells can be obtained through one-time sequencing, the single cell sequencing throughput is greatly improved, and the development of the single cell high-throughput chromatin transposase accessibility sequencing technology based on the microplate is actively promoted.

Description

Ultrahigh-throughput single-cell chromatin transposase accessibility sequencing method

Technical Field

The invention relates to the technical field of single cell sequencing, in particular to an ultra-high-throughput single cell chromatin transposase accessibility sequencing method.

Background

In the genome, most of the chromatin is tightly wound in the nucleus and has no transcriptional activity. Chromatin state is dynamically regulated in a cell-type specific manner, where partially compact chromatin becomes loose, which is called open chromatin (open chromatin) or accessible chromatin (accessible chromatin). The opening condition of cellular chromatin is detected, and transcription regulation information of the cell can be obtained, such as the position of the transcription factor capable of combining with the gene promoter, which genes of the cell are possible to be transcribed with high efficiency, and the like. Commonly used detection means are ATAC-seq, DNase-seq, MNase-seq, FAIRE-seq, ChIP-seq and the like, which are based on different principles for breaking and marking open areas of the chromatin, wherein, ATAC-seq (high-throughput chromatin transposase accessibility sequencing method, assay for transposase-accessible chromoprotein with high-throughput sequencing) uses modified Tn5 transposase, can randomly insert a specific DNA sequence as a transposon into an open region of chromatin, can directly capture the sequence of the whole open region completely, ATAC-seq is now widely used in open Chromosome sequencing (Agbleke AA, et. al., Advances in Chromosome and Chromosome Research: Perspectives from Multiple fields. mol cell.2020Sep 17; 79 (6): 881-901. doi: 10.1016/j. molcel.2020.07.003. Epub. 2020Aug 7. PMID: 32768408; PMCID: 7888594.).

Since 2009 Tom-Rich proposed Single-cell transcriptome sequencing technologies, various platforms were developed for high throughput Single-cell transcriptome sequencing with single-cell RNA sequencing cell Mol Immunol.2019 Mar; 16 (3): 242-249. doi: 10.1038/s 41423-019. PMC 0214-4.Epub 2019 Feb 22. PMID: 30796351; PMC6460502.), including microfluidic systems (Drop-Seq, inDrop-Seq and 10 Xnogenics), micro-fluidic systems (Microwell-Seq, Seq-well and BD Rhapody), cell/cell nucleus-pool systems (sci-RNA-Seq, sci-RNA-Seq and SPT 3). The accumulation of these technology platforms has also driven the development of Single cell ATAC technology (Sinha S, et. al., Profiling chromatography access at Single-cell resolution. genomics protocols bioinformatics.2021 Feb 10: S1672-0229(21)00011-5. doi: 10.1016/j.gpb.2020.06.010.Epub ahead of print. PMID: 33581341.) including Single cell ATAC based on microfluidic systems (10 XGenomics), μ ATAC-seq based on microwells (ICELL8), sci-ATAC-seq and sci-ATAC-seq3 of cell/cell nuclear split-pool systems. The single-cell ATAC platform based on micro-fluidic and micro-pore has high sensitivity, low pollution rate, low analysis flux, only about 10000 single-cell samples can be processed in a single experiment, and simultaneously, the cost is high. sci-ATAC-seq is limited by the number of split-pool combinations, and the analyzed nuclear flux is low and the intercellular contamination rate is high. On the basis of the former, the sci-ATAC-seq3 adopts a transposase without a label to insert a transposon fragment, and then adds a cell label by using a continuous two-round split-pool connection method, so that the combination number is increased, but partial sensitivity is sacrificed due to the efficiency problem of two-round connection in a nucleus. Meanwhile, the cell nucleus pre-labeling system needs multiple rounds of split-pool processes, so that the cell recovery efficiency is low (about 10%).

Disclosure of Invention

The invention provides an ultra-high flux single cell chromatin transposase accessibility sequencing method aiming at the defects in the prior art.

An ultra-high throughput single cell chromatin transposase accessibility 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 specific molecular tag transposase embedding complex comprising Tn5 transposase and a specific molecular tag sequence, wherein the specific molecular tag sequence comprises, 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 each sequence in the constructed sequencing library is derived;

a Mosaic endis sequence for binding to Tn5 transposase, said Mosaic endis sequence being a double-stranded structure in which one strand is linked to a second cell tag sequence;

c) bridging primer, which is used for connecting the molecular marker sequence in the a) and the specific molecular label sequence in the b), wherein the two ends of the bridging primer are provided with sequences which are respectively complementary and matched with the first bridging sequence and the second bridging sequence;

(2) taking a cell sample to be sequenced, and extracting cell nucleuses;

(3) adding a specific molecular tag transposase embedding compound into the cell nucleus extracted in the step (2) to perform transposition reaction;

(4) enabling a molecule labeling microbead and one or more cell nuclei to be in a separated space through a microplate technology or a microfluidic technology after transposition reaction, cracking the cell nuclei 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 molecule labeling sequence-specific molecule labeling sequence-chromatin transposase accessible genome sequence coupled with the microbead;

(5) collecting the microbeads coupled with the molecular marker sequence-specific molecular tag sequence-chromatin transposase accessibility genome sequence, and carrying out PCR amplification to obtain the chromatin transposase accessibility genome sequence with a first cell tag sequence, a second cell tag sequence and the specific molecular tag sequence;

(6) constructing a chromatin accessibility sequencing library by using the products obtained in the step (5), and then carrying out high-throughput sequencing to obtain chromatin transposase accessibility genome sequence 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 brought into a corresponding sequence of a genome during the transposition reaction of Tn5 transposase, so that under the condition that one molecular marker microbead is combined with a plurality of cells, the second cell tag sequence can be used for distinguishing, and if the two molecular marker microbeads are combined with a plurality of cells, the sequences of a plurality of cell sources combined with the same molecular marker microbead cannot be distinguished only by the first cell tag sequence on the molecular marker 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 replaces hydroxyl at 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 microbead is coupled with the amino through condensation of the carboxyl. 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 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 mode 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 the synthesis is random;

the specific molecular tag sequence is as follows: 5 '-ACACTCTTTCCCTACACGACGNNNNNNNNNNAGATGTGTATAAGAGACAG-3', wherein N represents any one of A/T/C/G, and the sequence of the Mosaic Ends which are complementary to form a double chain is as follows: 5'-CTGTCTCTTATACACATCT-3', respectively;

the bridging primer is: 5'-CGTCGTGTAGGGAAAGAGTGTGACGCTGCCGACGA-3' are provided.

Preferably, in step (3), one transposase complex in the specific molecular tag transposase embedding complex carries two gene segments, both of which are the specific molecular tag sequences, or one of which is the specific molecular tag sequence and the other of which is a universal sequence, wherein the universal sequence includes:

a primer binding sequence for amplification as a primer binding region in PCR amplification;

a Mosaic endis sequence for binding to Tn5 transposase, said Mosaic endis sequence being a double-stranded structure in which one strand is linked to a primer binding sequence for amplification.

Preferably, the cell sample to be sequenced contains 2 or more than 2 cells. The method for sequencing the accessibility of the single-cell chromatin transposase can realize the simultaneous sequencing of various cells.

Preferably, the bead bodies are magnetic beads,

in the step (4), the cell nuclei after transposition reaction 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 cell nuclei;

in the step (4), the hole dropping rate of the cell nucleuses 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 ultra-high-flux single-cell chromatin transposase accessibility 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 microplate method is used, the falling-hole rate can be greatly improved when the cells are added into the microplate.

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. For example, the micropores are cylindrical, wherein the depth of the micropores is 60 μm, the diameter of the micropores is 50 μm, and the hole pitch is 70 μm; the diameter of the bead body is 45 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 to obtain the microporous plate.

The accessibility sequencing method of the ultra-high-flux single-cell chromatin transposase is based on the accessibility sequencing method of the molecular marker micro-beads and the fixed cell nucleus by inserting the Tn5 into the high-flux single-cell chromatin transposase with the molecular marker transposon, and the platform can detect the specific genome openness information of millions of single cells at one time, thereby greatly improving the flux of single cell sequencing and actively promoting the development of the accessibility sequencing technology of the single-cell high-flux chromatin transposase based on a microporous plate.

Drawings

FIG. 1 is a flow chart of preparation of magnetic beads with molecular markers.

FIG. 2 shows the linker sequences and Tn5 transposase for linker embedding, wherein the two linker sequences respectively comprise specific molecular tag sequences and universal sequences, the linker sequences generate Tn5 embedded complexes through incubation with Tn5 naked enzyme, and a single Tn5 embedded complex simultaneously carries the specific molecular tag sequences and the universal sequences.

FIG. 3 is a schematic diagram of cells falling into a microplate, wherein each well can fall multiple nuclei, increasing the throughput of detecting cells, and cellular DNA fragments in the same well are distinguished by cell tag 4 on Tn 5.

FIG. 4 is a flow chart for constructing an ATAC library, 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 of FIG. 1; the bridging sequences include bridging sequence 1 and bridging sequence 2.

FIG. 5 is a diagram showing the size distribution of fragments of the prepared gene sequencing library.

FIG. 6 is a comparison of human and mouse mixed cell populations.

FIG. 7 is a DNA fragment size distribution diagram of a human and mouse sample.

FIG. 8 is a graph of peak annotation distribution for human and mouse samples.

FIG. 9 is a graph of mouse TSS enrichment.

FIG. 10 is an overlay distribution of mouse single cell ATAC and population ATAC peak.

FIG. 11 is a graph comparing the distribution of ATAC of mouse single cell and ATAC read of population on chromosome 8 at 0-25 Mbp.

FIG. 12 is a flow chart of the construction of ATAC library by Tn5 embedding method with both ends labeled, 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 in FIG. 1.

Detailed Description

The present invention will be further described with reference to the following examples, which are intended to illustrate the present invention and not to limit the scope of the present invention, and all simple modifications of the preparation method of the present invention based on the idea of the present invention are within the scope of the present invention. The following examples are experimental methods without specifying specific conditions, and generally follow the methods known in the art. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

The reagents used were not described in any concentration ratio by volume.

Example 1: microplate preparation

The microplate size (well plate size 1.8cm × 1.8cm) was designed on an experimental scale (50 ten thousand each of human 293T cells and mouse 3T3 cells), and micropores of a cylindrical shape having a micropore depth of 60 μm, a micropore diameter of 50 μm, and a pore spacing of 70 μm were etched on a silicon wafer as an initial mold. And then, pouring Polydimethylsiloxane (PDMS) on the silicon chip, taking off the PDMS after molding to form a second mold with the microcolumns on the plate, wherein the micropore plate used in the final experiment is agarose (prepared by non-enzyme water) with the concentration of 5% (mass ratio), pouring the hot melt agarose on the PDMS microcolumn plate for condensation molding, and taking off the agarose plate at the moment to form the micropore plate with a certain thickness. When in storage, the DPBS-EDTA mixed solution harmless to cells is added, and the mixture is covered and stored in a refrigerator at 4 ℃, so that the good working state of the microporous plate can be ensured.

Example 2: preparation of molecular marker magnetic beads

Magnetic beads were purchased from Suzhou knoyi microsphere science, Inc. (CatCOOH-20190725), surface carboxyl-coated, 45 μm in diameter. The preparation process of the molecular marker magnetic beads is shown in figure 1, and comprises 5 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 sequences of the various segments are shown in Table 1 below.

TABLE 1

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 4⁶And (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 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 molecular marker sequences were as follows:

(5) the labeled magnetic beads are annealed to the bridging primer. The magnetic beads were mixed with a bridging primer (bridging primer sequence: 5'-CGTCGTGTAGGGAAAGAGTGTGACGCTGCCGACGA-3') and gradient annealed to form cohesive ends. Sequences not bound to the bridging primer were excised using exonuclease EXO I. After the excision was complete, the column was washed once with 150. mu.l of TE-SDS, TE-TW and finally resuspended in TE-TW and stored at 4 ℃ until use.

Example 3: preparation of specific molecular tag transposase embedding complex

Tn5 transposase naked enzyme was purchased from Nanjing Novozam Biotech Ltd. Transposase and embedding buffer were supplied from the kit (Vazyme) Tn5 Transposome (S111) manufactured by Nanjing Novozam Biotech, Inc.

(1) The embedded nucleic acid sequences are shown in Table 2 below.

TABLE 2

Wherein the specific tag fragment comprises a specific molecular tag 10 XN which is the core sequence of a cell tag sequence, the core sequence corresponding to each Tn5 complex is different, and the 10 XN sequence can have 4 due to 4 selections of A/T/C/G at each site¹⁰And (4) selecting. N represents any of A/T/C/G, and is randomly synthesized.

(2)96 annealing with specific molecular tag oligonucleotide sequence (P7 Adapter fragment + molecular Ends fragment annealing to get general sequence, i.e. P7 Adapter-molecular Ends/molecular Ends in the linker in FIG. 2; specific tag fragment + molecular Ends fragment annealing to get specific molecular tag sequence, i.e. bridging sequence 2-cell tag 4-molecular Ends/molecular Ends in FIG. 2). Each well of 96 wells contained two embedded fragments, including the same P7 adapter fragment + molecular Ends fragment between wells and a specific tag fragment + molecular Ends fragment specific between wells.

(3) Uniformly adding Tn5 naked enzyme and embedding buffer solution into a 96-well plate, then adding 96 label joint freezing solutions in the step (2) into each well, blowing and beating for 5 times, incubating for 1h at 30 ℃, and storing in a refrigerator at-20 ℃ (figure 2).

Example 4: fresh tissue cell nucleus extraction

50mg of fresh tissue sample was pulverized with liquid nitrogen and quickly transferred to a pre-cooled 1.5ml EP tube. With 1ml of lysis buffer (ddH)₂O，10mM Tris-HCL pH7.4，10mM NaCl，3mM MgCl ₂1% BSA, 0.1% Tween-20, 0.1% IGEPAL CA-630, 0.01% digitonin) were resuspended and lysed on ice for 3 min. Lysis was followed by RSBT buffer (ddH)₂O，10mM Tris-HCl pH7.4，10mM NaCl，3mM MgCl ₂1% BSA, 0.1% Tween-20) and filtered to remove tissue debris. Nuclei were fixed with 1% formaldehyde for 10 min at room temperature. The fixation cross-linking is terminated with glycine. Preparing frozen stock solution (ddH)₂O, 50mM Tris-HCl pH8.0, 25% glycerol, 5mM Mg (OAc)₂，0.1mMEDTA), adding 5. mu.l of 5mM DTT and 20. mu.l of 50 × protease inhibitor cocktail into 975. mu.l of the frozen stock solution, and carrying out-80 freezing after cell nucleus is resuspended. Or suspending cell nuclei with 1ml of RSBT buffer, and counting for later use. When the frozen sample is recovered, the sample is taken out, placed in an oven at 37 ℃ for unfreezing for 2min, centrifuged at 500g/5min, the supernatant is discarded, 200 mu l of lysate is used for resuspension, and the frozen sample is placed on ice for 3 min. After washing and filtering with RSBT, counting for standby.

Example 5: extraction of cell nucleus from mixed cell of human 293T and mouse 3T3

Mouse fibroblasts (3T3) and human embryonic kidney cells (293T) were mixed in 200 ten thousand each, washed once with PBS, transferred to a 1.5ml EP tube, and then lysed with 1ml of lysis buffer (ddH)₂O，10mM Tris-HCl pH7.4，10mM NaCl，3mM MgCl ₂1% BSA, 0.1% Tween-20, 0.1% IGEPAL CA-630, 0.01% digitonin) were resuspended and lysed on ice for 3 min. Lysis was followed by RSBT buffer (ddH)₂0，10mM Tris-HCl pH7.4，10mM NaCl，3mM MgCl ₂1% BSA, 0.1% Tween-20) were washed twice. Nuclei were fixed with 1% formaldehyde for 10 min at room temperature. The fixation cross-linking is terminated with glycine. Preparing frozen stock solution (ddH)₂O, 50mM Tris-HCl pH8.0, 25% glycerol, 5mM Mg (OAc)₂0.1mM EDTA), adding 5. mu.l of 5mM DTT and 20. mu.l of 50 × protease inhibitor cocktail to 975. mu.l of the frozen stock solution, and carrying out-80 freezing after cell nucleus is resuspended. Or suspending cell nuclei with 1ml of RSBT buffer, and counting for later use. When the frozen sample is recovered, the sample is taken out, placed in an oven at 37 ℃ for unfreezing for 2min, centrifuged at 500g/5min, the supernatant is discarded, 200 mu l of lysate is used for resuspension, and the frozen sample is placed on ice for 3 min. After washing and filtering with RSBT, counting for standby.

Example 6: tn5 pre-labeling of mixed human 293T and murine 3T3 cells

The nuclei prepared in example 5 were taken, resuspended, filtered and counted using 1ml of RSBT. Preparation of 2 XTD buffer (ddH)₂O，20mM Tris-HCl pH7.6，10mM MgCl₂20% dimethylformamide), preparing a phasing buffer (22.5. mu.l per well, including 12.5. mu.l of 2 XTD buffer, 9.5. mu.l of 1 XDPBS, 0.25. mu.l of 1% digitonin, 0.25. mu.l of 10% Tween-20), resuspending the nuclei in the phasing buffer into 96-well plates, approximately 1 million nuclei per well. Working examplesThe transposase-embedded complex in 3 was added to a 96-well plate containing nuclei at 2.5. mu.l per well, and reacted at 55 ℃ for 30 min. After completion of the reaction, 25. mu.l of 2 Xstop solution (25ml of 40mM EDTA, 3.9. mu.l of 6.4M Spermidine) was added to each well, and the mixture was allowed to stand at 37 ℃ for 15 min. The collected liquid was mixed into a 15ml centrifuge tube, 500g/5min, the supernatant was discarded, and 1ml RSBT was washed once. Counting, cell nuclei were dispensed into 1.5ml EP tubes, 50 ten thousand nuclei per tube, 20. mu.l RSBT resuspended, and 30. mu.l PNK reaction solution (5. mu.l 10 XPNK buffer, 5. mu.l 10mM ATP, 10. mu.l ddH) added₂O, 10. mu.l PNK enzyme), incubated at 37 ℃ for 30min, centrifuged after the reaction is complete, and the supernatant is discarded. RSBT was washed twice and then placed on ice for use.

Example 7: microplate ATAC sequencing of human 293T and murine 3T3 Mixed cells

Tn5 was used to label the nuclei in example 6. The cell nucleus suspension is dripped into a microplate, 80-90% of the cell nuclei fall into the micropores by using a centrifuge for short centrifugation, 0-10 cell nuclei are captured in each hole (see figure 3), and the cellular DNA fragments in the same hole are distinguished by the cell label 4 on Tn 5. Annealing the bridging primer and the labeled magnetic beads to form sticky ends on the fragments on the labeled magnetic beads. Adding 20 ten thousand molecular marker magnetic beads into the microplate with the cell nucleus, placing the microplate on a magnet, gently mixing the magnetic beads uniformly to enable the magnetic beads to cover more than 99% of micropores, and washing away redundant molecular marker magnetic beads by using RSBT solution. mu.L of lysate (100. mu.l of 10% SDS, 40. mu.l proteinase K, 100. mu.l of 10 XT 4 buffer, 200. mu.l of 50% PEG8000 (in terms of mass/volume), 560. mu.l of 10mM Tris-HCl pH8.0) was slowly dropped into the microplate filled with the magnetic beads, and the lysate was incubated for 30 minutes at room temperature for sufficient complementary hybridization between the bridge sequences on the magnetic beads and the bridge primers on the nuclei. After completion of incubation, the plate was inverted on a magnet, the magnetic beads with the molecular marker-DNA complexes were collected, transferred to a 1.5ml EP tube, washed twice with 6 XSSC, and then washed once more with 50mM Tris pH8.0, and 50. mu.L of the ligation mixture (2. mu. L T4 ligase, 5. mu. L T4 buffer, 2. mu.L dNTP, 10. mu.L of 50% PEG8000 (Mass to volume ratio), 31. mu.L of ddH was added to the EP tube containing the molecular marker magnetic beads₂O), and the reaction mixture was left at 25 ℃ for 1.5 hours. After the ligation reaction was completed, the magnetic beads were labeled with TE-SDS, TE-TW, 10mM Tris, pH8.0Each wash was performed with 100. mu.L extension (20. mu.L of 5 XTT buffer, 10. mu.L dNTP, 2.5. mu.L Klenow polymerase, 20. mu.l of 50% PEG (Mass to volume ratio) and 47.5. mu.l ddH)₂O) suspending the magnetic beads, and reacting at 37 ℃ for 1 hour. After the reaction, the labeled magnetic beads were washed once with TE-SDS, TE-TW, and 10mM Tris, pH8.0, respectively, on a magnetic rack, suspended with 500. mu.L of 0.1M NaOH, and incubated at room temperature for 5 minutes to make single strands. Then washed twice with TE-TW and once with 10mM Tris pH8.0, the target fragment was enriched from the beads using index P5 and index P7 primers, and the PCR product was purified using Novonza to obtain a product of 300-500 bp.

The detailed reaction scheme is shown in FIG. 4, and the library size distribution is around 300-500bp, as shown in FIG. 5.

Constructing a gene sequencing library by the method, and sequencing by using an MGI/Illumina next generation sequencer after the gene sequencing library is constructed. And (4) carrying out resolution, screening and comparison on data returned by sequencing to obtain a cell by peak matrix. 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 FIGS. 6-11, there was very little double-cell contamination (FIG. 6), the fragment size was as expected (FIG. 7), the peak distribution was normal (FIG. 8), TSS was enriched (FIG. 9), the mouse 3T3 cell population and the single-cell ATAC peak distribution (FIGS. 10 and 11), and the sequencing quality could reach the high-throughput sequencing level of single-cell ATAC.

The sequences of the primers used for the pooling are shown in Table 3 below. Wherein N represents any one of A/T/C/G, and is randomly synthesized. S represents a thio modification of the terminal base, and improves the stability of the terminal base.

TABLE 3

Example 8

The invention can also be applied to a single cell sequencing platform based on microfluidics. After obtaining a fixed cell nucleus with one round of cell labeling by the method described in example 6, a 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 bridging primer), inlet 2 injected with 40. mu.l of single-cell ATAC gel microbeads containing lysate (10 × Genomics #2000132, the microbead sequence as described in example 1), and inlet 3 injected with 240. mu.l of Particioning Oil (10 × Genomics # 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 for exo-, single-, double-stranded synthesis reactions according to the protocol described in example 7, and finally the gene sequencing library was obtained by PCR amplification and purification, sequencing and signal generation analysis. The gene sequencing library was sequenced and analyzed for information according to the procedure in example 7.

Example 9

The invention can also be applied to the Tn5 embedding method with both ends labeled.

Embedding was carried out according to the method described in example 3, but in the embedding, 1 embedded fragment was contained in each well of 96 wells, which was specific tag fragment + molecular Ends fragment between wells, and specific tag fragment + molecular Ends fragment annealed to obtain specific molecular tag sequence, i.e., bridging sequence 2-cell tag 4-molecular Ends/molecular Ends in FIG. 2.

Then, the nuclei were labeled according to the protocol of example 6, and the labeled nuclei were captured, ligated, and extended in a microplate according to example 7. The DNA fragment was single-stranded by adding 500. mu.L of 0.1M NaOH solution for 5 minutes, and then extended by adding P7 adapter-molecular Ends primer (i.e., primer including the sequences of P7 adapter and molecular Ends fragments). After the extension is completed, the product of the top chain is collected by high temperature denaturation, and the magnetic beads are removed. The gene sequencing library was obtained by PCR amplification and purification using index P5 and index P7 primers (see FIG. 12). The gene sequencing library was sequenced and analyzed for information according to the procedure in example 7.

Sequence listing

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<220>

<221> misc_feature

<222> (16)..(21)

<223> n is a, t, c or g.

<400> 2

cgatcgtgtc accgannnnn nccctgtagt gagtcg 36

<210> 3

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (15)..(20)

<223> n is a, t, c or g.

<400> 3

gacgctgccg acgannnnnn cgatcgtgtc accga 35

<210> 4

<211> 109

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (48)..(53)

<223> n is a, t, c or g.

<220>

<221> misc_feature

<222> (69)..(74)

<223> n is a, t, c or g.

<220>

<221> misc_feature

<222> (90)..(95)

<223> n is a, t, c or g.

<400> 4

tttagggata acagggtaat aagcagtggt atcaacgcag agtacgtnnn nnncgactca 60

ctacagggnn nnnntcggtg acacgatcgn nnnnntcgtc ggcagcgtc 109

<210> 5

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

cgtcgtgtag ggaaagagtg tgacgctgcc gacga 35

<210> 6

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gtctcgtggg ctcggagatg tgtataagag acag 34

<210> 7

<211> 50

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (22)..(31)

<223> n is a, t, c or g.

<400> 7

acactctttc cctacacgac gnnnnnnnnn nagatgtgta taagagacag 50

<210> 8

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

ctgtctctta tacacatct 19

<210> 9

<211> 63

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gaacgacatg gctacgatcc gacttgcctg tccgcggaag cagtggtatc aacgcagagt 60

acg 63

<210> 10

<211> 50

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (26)..(35)

<223> n is a, t, c or g.

<400> 10

tgtgagccaa ggagttgttg tcttcnnnnn nnnnngtctc gtgggctcgg 50

Claims (4)

1. An ultra-high throughput single cell chromatin transposase accessibility 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 specific molecular tag transposase embedding complex comprising Tn5 transposase and a specific molecular tag sequence, wherein the specific molecular tag sequence comprises, 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 each sequence in the constructed sequencing library is derived;

a Mosaic endis sequence for binding to Tn5 transposase, said Mosaic endis sequence being a double-stranded structure in which one strand is linked to a second cell tag sequence;

c) bridging primer, which is used for connecting the molecular marker sequence in the a) and the specific molecular label sequence in the b), wherein the two ends of the bridging primer are provided with sequences which are respectively complementary and matched with the first bridging sequence and the second bridging sequence;

(2) taking a cell sample to be sequenced, and extracting cell nucleuses;

(3) adding a specific molecular tag transposase embedding compound into the cell nucleus extracted in the step (2) to perform transposition reaction;

in step (3), one transposase complex in the specific molecular tag transposase embedding complex carries two gene segments, both of which are the specific molecular tag sequences, or one of which is the specific molecular tag sequence and the other of which is a universal sequence, wherein the universal sequence comprises:

a primer binding sequence for amplification as a primer binding region in PCR amplification;

a Mosaic Ends sequence for binding Tn5 transposase, said Mosaic Ends sequence having a double-stranded structure wherein one strand is linked to a primer binding sequence for amplification;

(4) enabling a molecule labeling microbead and one or more cell nuclei to be in a separated space through a microplate technology or a microfluidic technology after transposition reaction, cracking the cell nuclei 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 molecule labeling sequence-specific molecule labeling sequence-chromatin transposase accessible genome sequence coupled with the microbead;

(5) collecting the microbeads coupled with the molecular marker sequence-specific molecular tag sequence-chromatin transposase accessibility genome sequence, and carrying out PCR amplification to obtain the chromatin transposase accessibility genome sequence with a first cell tag sequence, a second cell tag sequence and the specific molecular tag sequence;

(6) constructing a chromatin accessibility sequencing library by using the product obtained in the step (5), then carrying out high-throughput sequencing to obtain specific genome openness information of millions of single cells,

the first cell label sequence comprises a plurality of specific segments, the second cell label sequence comprises at least one specific segment, the specific segments at different positions are selected from the same or different specific segment libraries, the first cell label sequence and the second cell label sequence utilize the arrangement combination mode of the specific segments to identify cells differently,

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, 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,

the molecular marker sequence is as follows: 5 '-TTTAGGGATAACAGGGTAATAAGCAGTGGTATCAAC GCAGAGTACGTNNNNNNCGACTCACTACAGGGNNNNNNTCGGTGACACGATCG NNNNNNTCGTCGGCAGCGTC-3', wherein N represents any one of A/T/C/G, and the synthesis is random;

the specific molecular tag sequence is as follows: 5 '-ACACTCTTTCCCTACACGACGNNNNNNNNNN AGATGTGTATAAGAGACAG-3', wherein N represents any one of A/T/C/G, and the sequence of the Mosaic Ends which are complementary to form a double chain is as follows: 5'-CTGTCTCTTATACACATCT-3', respectively;

the bridging primer is: 5'-CGTCGTGTAGGGAAAGAGTGTGACGCTGCCGACGA-3' the flow of the air in the air conditioner,

the cell sample to be sequenced contains 2 or more than 2 cells,

the bead body is a magnetic bead,

in the step (4), the cell nuclei after transposition reaction 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 cell nuclei;

in the step (4), the hole dropping rate of the cell nucleuses 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.

2. The method for sequencing by ultra high throughput single cell chromatin transposase accessibility of claim 1, wherein said microbeads are coupled to molecular marker sequences in a manner that: amino replaces hydroxyl at 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 microbead is coupled with the amino through condensation of the carboxyl.

3. The method for sequencing the accessibility of a single cell chromatin transposase of ultra-high throughput of claim 1, wherein the depth of the microwell in the microwell plate is 30-160 μ ι η, the diameter of the microwell is 20-150 μ ι η; the diameter of the bead body is 20-145 μm.

4. The method for sequencing the accessibility of the transposase enzyme for ultra-high throughput single-cell chromatin of claim 3, wherein the microplate is prepared by:

(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.

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