CN115537408A - Single cell multi-omics library and construction method thereof - Google Patents
Single cell multi-omics library and construction method thereof Download PDFInfo
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Abstract
The invention relates to the technical field of biology, in particular to a single-cell multi-component library and a construction method thereof. The sequencing joint at one end of the Tn5 transposon is transformed into a specific connecting sequence for connecting barcode; can complete the simultaneous library establishment of the transcriptome and chromatin open areas of single cells, adopts a cell combination labeling technology, does not need additional instruments and equipment, and greatly reduces the library establishment cost compared with the scheme of 10xGenomics company. The library structure adopted by the invention is adapted to the conventional general PE150 sequencing for the second-generation sequencing, a self-defined sequencing program is not needed, and the sequencing cost can be greatly reduced compared with SHARE-seq.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a single-cell multi-component library and a construction method thereof.
Background
There are currently two main approaches to single cell sequencing: one is to adopt micro-flow control, micro-porous plate and other modes to carry out single cell isolation in space, and the other is to distinguish single cells by combining label marks based on a cell label technology. Most commercialized single cell sequencing technologies are based on a spatial isolation mode, for example, a single cell library construction scheme of 10x Genomics, which has the advantages of simple operation, capability of being accessed to a 10x library construction process only by completing single cell suspension preparation work by researchers, low cell throughput, capability of detecting only about 10000 cells in a single library construction, and high price.
The combinatorial cell tagging technique does not require spatial single cell separation, but rather labels unique combinatorial tags on each cell band by multiple random barcode tags. Single cell sequencing technologies currently developed using combinatorial tagging techniques include sci-RNA-seq, sci-CAR, sci-ATAC-seq, SPLIT-seq, paired-seq, SHARE-seq, and the like. Taking SPLiT-seq as an example, the method is performed by dispersing the reverse transcribed cells into 96-well plates, each well having a unique barcode attached to the cDNA, then collecting the cells, and performing the second and third rounds of barcode attachment in the same manner, so that each cell has a unique combination of barcodes for distinguishing from other cells.
The sci-CAR is the earliest to complete the single cell multiomic library establishment through a combined label technology, namely mRNA and chromatin development regions in single cells can be detected simultaneously, but the sci-CAR adopts a small number of combined labels, so the detected cell flux is low.
Paired-seq is a single-cell multigroup technology developed by any army team at the university of California, the detection flux can be increased to 10^6 by 4 rounds of barcode labeling, the technology firstly transposes a cell nucleus by using Tn5 transposase with barcode and an enzyme digestion site, then carries out reverse transcription reaction, simultaneously connects three rounds of barcode on cDNA and Tn5 labeled DNA, and separates and sequences a library by enzyme digestion after the library is amplified (see figure 1).
SHARE-seq is a single-cell multiomic sequencing technology developed by Jason D.Buenrostro and AvivRegev team of the Massachusetts institute of technology, which changes Tn5 transposase conditions and library isolation conditions compared to Paired-seq and can detect mRNA in cytoplasm, and the technology transposes with Nextera Tn5 transposase first, then carries out reverse transcription with biotin-modified primers, carries out decrosslinking after three rounds of barcode connection, separates library with streptavidin magnetic beads, and sequences (see FIG. 2).
Paired-seq can not detect mRNA in cytoplasm, and 8 Tn5 transposases need to be constructed, so that the technical threshold is high. Two Tn5 transposases need to be constructed for SHARE-seq, mRNA in cytoplasm can be detected, the data quality is higher than that of Paired-seq, but the library structure of SHARE-seq is not suitable for a PE150 sequencing method commonly used by the existing sequencing company, a custom sequencing program is needed, and the sequencing price of the domestic sequencing company is very expensive, so that the sequencing cost of the SHARE-seq library is very high.
Disclosure of Invention
In view of the above, the present invention provides a single-cell multi-omic library and a method for constructing the same. The method reconstructs the Tn5 insertion sequence for barcode connection, the library structure is similar to SPLiT-seq and Paired-seq, the method is suitable for PE150 sequencing, and the sequencing cost is greatly reduced compared with SHARE-seq.
In order to achieve the above object, the present invention provides the following technical solutions:
a Tn5 transposase complex, designated Tn5-2, comprising a Tn5 transposase and a first assembly sequence;
the nucleotide sequence of the first assembly sequence is shown as SEQ ID NO.1 and SEQ ID NO. 2.
In some embodiments, the Tn5 transposase complex further comprises a second assembly sequence, designated Tn5-1, comprising a Tn5 transposase, the first assembly sequence, and the second assembly sequence;
the nucleotide sequence of the second assembly sequence is shown as SEQ ID NO.1 and SEQ ID NO. 3.
In some embodiments, the sequence set forth in SEQ ID No.1 has a phosphorylation modification at the 5 'end and a dideoxy modification at the 3' end; the 5' end of the sequence shown in SEQ ID NO.3 has phosphorylation modification.
The invention also provides a method for constructing the single-cell multi-omic library, which comprises the following steps:
(1) Fixing the cells, and performing permeabilization treatment;
(2) Transposing the cells treated in the step (1) by using the Tn5-1 transposase complex to obtain transposed cells; the transposed cells comprise labeled chromatin opening regions and unlabeled mRNA;
(3) Reverse transcription of mRNA in the cells obtained in step (2) by using a reverse transcription primer; the reverse transcription primer sequence is shown as SEQ ID NO.295.
(4) Sequentially carrying out a first round, a second round and a third round of Barcode labeling on the reverse transcription product in the step (3) by using primers;
(5) Collecting the cells marked in the step (4), performing crosslinking release, separating cDNA (complementary deoxyribonucleic acid) and chromatin by using streptavidin magnetic beads, and respectively building libraries to obtain an RNA (ribonucleic acid) library and an ATAC (atom transfer acetic acid) library;
in the construction process of the RNA library, the Tn5-1 transposase compound is used for fragmenting cDNA;
in the present invention, in the step (1), the fixing agent used for fixing the cells is not particularly limited, and may be any one of the types commonly used in the art; in some embodiments, the fixative for fixation is formaldehyde or paraformaldehyde. In the present embodiment, the treatment agent for permeabilization is 0.1% NP40,0.1% Triton X-100,0.01 to 0.05% Digitonin or the like, and is suitable for various cell types. The cells after the permeabilization treatment can be passed through a reagent.
In some embodiments, in step (2), the reverse transcribed primer is modified with a biotin. The invention utilizes biotin to modify reverse transcription primer, carries out intracellular reverse transcription, adds streptavidin magnetic bead into the reverse transcription product, combines cDNA modified with biotin with the streptavidin magnetic bead, and separates ATAC library from cDNA library by capturing the magnetic bead.
In the step (4) of the invention, barcode primers are used for labeling the cells obtained after the reverse transcription reaction in the step (3), 96 labels are provided for each round, after three rounds of Barcode labeling, 96 × 96=884,736 combined labels can be generated, each cell carries one combined label, and the combined label is distinguished from other cells.
Specifically, the first Round of Barcode labeled primers comprise Round1linker and Round1 Barcode; the second Round of Barcode labeled primers comprise Round2 linker and Round2 Barcode; the third Round of Barcode labeled primers comprises Round3linker and Round3 Barcode;
the Round1 Barcode sequentially comprises a 5' phosphorylation label, a sequence which is complementary with a Round2 linker part base of the second Round Barcode label, a first Round Barcode and a sequence which is complementary with a Round1linker part base of the first Round Barcode label from 5' to 3 ';
the Round2 Barcode sequentially comprises a 5' phosphorylation marker, a sequence complementary to a part of bases of Round3linker marked by the third Round of Barcode, a second Round of Barcode and a sequence complementary to a part of bases of Round2 linker marked by the second Round of Barcode from 5' to 3 ';
the Round 3Barcode sequentially comprises a sequence complementary to an upstream primer amplified from the cDNA library, UMI, the third Round of Barcode and a sequence complementary to a part of bases of Round3linker labeled with the third Round of Barcode from 5 'to 3'.
In some embodiments, the nucleotide sequence of the Round1linker is shown as SEQ ID NO.289, and the nucleotide sequence of the Round1 barcode is shown as SEQ ID NO. 1-96;
the nucleotide sequence of the Round2 linker is shown as SEQ ID NO.290, and the nucleotide sequence of the Round2 barcode is shown as SEQ ID NO. 97-192;
the nucleotide sequence of Round3linker is shown in SEQ ID NO.291, and the nucleotide sequence of Round 3barcode is shown in SEQ ID NO. 193-288.
In the invention, in the nucleotide sequence of Round1 Barcode, 8 bases behind a sequence which is complementary with partial bases of Round2 linker marked by the second Round Barcode are first Round Barcode; in the nucleotide sequence of Round2 Barcode, 8 bases following the sequence complementary to the partial base of Round3linker labeled with the third Round Barcode are the second Round Barcode; in the nucleotide sequence of Round 3Barcode, 8 bases after the UMI sequence is the third Round of Barcode.
In some embodiments, in step (5), the agent used for de-crosslinking comprises: 2 × RCB, proteinase K and RI;
the 2 × RCB comprises the following components: 1M Tris 8.0, 5MNaCl, 10% SDS and nucleic-Free Water (NFw).
In some embodiments, the step (5) specifically comprises:
(a) Collecting the cells marked in the step (4), and performing crosslinking release; separating the product of the de-crosslinking by streptavidin magnetic beads to obtain cDNA and chromatin;
(b) Purifying the chromatin obtained by the separation in the step (a), and then carrying out PCR amplification, enrichment and purification to obtain an ATAC library;
(c) Taking cDNA obtained by separation in the step (a), pre-amplifying, purifying, and adding the Tn5-2 transposase compound to obtain a Tag-cDNA purified product;
and carrying out PCR amplification, enrichment and purification on the purified product of the Tag-cDNA to obtain an RNA library.
The invention also provides libraries obtained by the construction method described above, including RNA libraries and ATAC libraries. The obtained library is compared with a 10x platform or a SHARE-seq, and the result shows that the quality of the library constructed by the invention is comparable to that of the 10x platform or the SHARE-seq.
On the basis of SHARE-seq, the invention modifies the library structure, modifies the sequencing joint at one end of Tn5 transposon into a specific connecting sequence for connecting barcode; can complete the simultaneous library establishment of the transcriptome and chromatin open areas of single cells, adopts a cell combination labeling technology, does not need additional instruments and equipment, and greatly reduces the library establishment cost compared with the scheme of 10xGenomics company. The library structure adopted by the invention is adapted to the conventional general PE150 sequencing for second-generation sequencing, a self-defined sequencing program is not needed, and the sequencing cost can be greatly reduced compared with SHARE-seq.
Drawings
FIG. 1 is a schematic diagram illustrating Paired-seq library construction;
FIG. 2 is a schematic diagram showing SHARE-seq library construction
FIG. 3 is a schematic flow diagram illustrating the construction of a single-cell multiomic library of the present invention;
FIG. 4 illustrates ATAC detection of valid fragments versus other platform data in accordance with the present invention;
FIG. 5 illustrates the TSS enrichment of ATAC data of the present invention compared to other platform data;
FIG. 6 shows effective UMI comparison with other platform data for RNA detection according to the present invention;
FIG. 7 shows the comparison of the number of effective genes detected by RNA of the present invention with the data of other platforms.
Detailed Description
The invention provides a single cell multi-omics library and a construction method thereof. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
Interpretation of terms:
single cell multinomic sequencing (single cell multi-omics): the single cell multigroup technology is developed based on a single cell library building technology and a high-throughput next-generation sequencing technology, and can simultaneously detect two or more kinds of omics information such as transcriptome, genome, epigenome, proteome or space state and the like at the single cell level.
Transcriptome sequencing (RNA-seq): the high-throughput sequencing of RNA transcribed from a gene of the whole genome of a specific cell in a certain functional state mainly comprises mRNA and non-coding RNA.
Chromatin accessibility sequencing (ATAC-seq): an open region is arranged between chromatin nucleosomes, a modified Tn5 transposase is used to design transposable DNA as a sequencing adaptor, and the sequencing adaptor is directly introduced into the open chromatin for sequencing.
Transposition: using the modified Tn5 transposase, specific sequences were inserted into the chromatin opening regions.
Reverse transcription: the process of synthesizing DNA using RNA as template, namely DNA synthesis under the guidance of RNA. In this process, the process of nucleic acid synthesis and transcription (DNA to RNA) is opposite to the flow of genetic information (RNA to DNA), and is called reverse transcription.
De-crosslinking of fixed cells: after cell fixation, the DNA cross-linked to the protein is released in the presence of high temperature, high salt, SDS and proteinase K.
DNA magnetic bead purification product: by utilizing the characteristic that DNA magnetic beads preferentially adsorb large fragments, the DNA with the size in a required range can be obtained by adsorbing the fragments with the specific size through the magnetic beads in a certain proportion.
Unless otherwise specified, the test materials used in the present invention are all common commercial products and are all available on the market.
The invention is further illustrated by the following examples:
example 1
The embodiment provides a method for constructing a single-cell omics library, a flow schematic diagram is shown in figure 3, and the method specifically comprises the following steps:
(1) Primer synthesis:
synthesizing Round1, round2, round 3barcode sequence and other primer sequences;
wherein, the nucleotide sequences of Round1_ 01-96 are shown as SEQ ID NO. 1-96 in sequence, the nucleotide sequences of Round2_ 01-96 are shown as SEQ ID NO. 97-192 in sequence, and the nucleotide sequences of Round3_ 01-96 are shown as SEQ ID NO. 193-288 in sequence.
TABLE 1 Round1 primer sequences
TABLE 2 Round2 primer sequences
TABLE 3 Round3 primer sequences
TABLE 4 other primer sequences
(2) Annealing of the primer:
1. the Round1, round2, round 3barcode primers were diluted with TE buffer (10 mM Tris-HCl 8.0,0.1mM EDTA) to 100uM, and the Round1, round2, round3linker were diluted with TE buffer to 1mM;
2. mixing the resulting mixture with a final concentration of 9uM Round1linker and 10uM Round1 barcode, and adding the mixture into a 96-well plate;
3. mixing the final concentration of 11uM Round2 linker and 12uM Round2 barcode, and adding into a 96-well plate;
4. mixing the final concentrations of 13uM Round3linker and 14uM Round3 barcode, and adding the mixture into a 96-well plate;
5. the 96-well plate was placed in PCR for annealing, the procedure was as follows:
TABLE 5
6. Barcode was dispensed into 96-well plates at a volume of 10ul per well and stored at-80 ℃.
(3) Construction of Tn 5:
1. synthesizing primers Tn5_ rev:/5 Phos/CTGTCTCTTATACACACAT/3 ddC/, tn5_ fw _ 1;
2. annealing of the primer: the following system was prepared in a PCR tube using an Annealing Buffer (Vazyme) to dissolve primers to 10 uM:
TABLE 6
3. Putting the mixture into a PCR instrument for annealing, and carrying out the following procedures:
TABLE 7
Hot lid | 105 |
|
75℃ | 15min | |
60 | 10min | |
50℃ | 10min | |
40 | 10min | |
25℃ | 30min |
4. Tn5-1 was constructed by adding the following components to the PCR tube:
TABLE 8
TruePrep Tagment Enzyme(Vazyme) | |
Adapter Mix | |
1 | 3.5 |
Adapter Mix | |
2 | 3.5μl |
Coupling Buffer | 39μl |
Total of | 50μl |
5. Tn5-2 was constructed by adding the following components to the PCR tube:
TABLE 9
TruePrep Tagment Enzyme(Vazyme) | |
Adapter Mix | |
1 | 7μl |
Coupling Buffer | 39μl |
Total of | 50μl |
6. Fully and uniformly blowing, putting into a PCR instrument, reacting for 1 hour at 30 ℃, and preserving at-20 ℃.
(4) Cell fixation:
first, a cell suspension was prepared, the cell suspension was centrifuged at 500g for 5min at room temperature, and the resulting cell pellet was resuspended with 4 ℃ pre-cooled DPBS-RI to a cell density of 1,000,000cells/ml. 1ml of DPBS-RI cell suspension was added with 5.4. Mu.l of 37% formaldehyde solution (final concentration: 0.2%) and fixed on ice for 10min. Then, the cells were centrifuged at 500g for 5min at 4 ℃ to remove the supernatant, thereby obtaining a fixed cell pellet.
DPBS-2RI | Volume(μl) |
1×DPBS | 4000 |
RNase Inhibitor(RI) | 10 |
(5) Cell lysis:
the fixed cell pellet was resuspended in 1ml of pre-cooled RSB-T-RI, and after uniform blowing, 10ul of NP40 (final concentration of 0.1%) was added, and again uniform blowing was carried out with a gun. And placed on ice to lyse the cells for 5min. Then, the mixture was centrifuged at 1000g for 5min at 4 ℃ to remove the supernatant. Resuspending the cell pellet with 1ml of precooled RSB-T-RI, passing through a 40 μm filter membrane after blowing uniformly, centrifuging for 5min at 4 ℃ at 1000g, removing the supernatant, collecting the pellet, resuspending the cell pellet with precooled 0.2ml of RSB-T-RI, taking 5 μ l of the cell pellet into a PCR tube after blowing uniformly, and adding 5 μ l of 0.2% of trawl blue for counting. Based on the cell concentration obtained by counting, the cells were diluted to 5,000cells/. Mu.l with RSB-T-RI.
TABLE 11
RSB-T-RI | Volume(μl) |
1×RSB-T | 4000 |
RNase Inhibitor(RI) | 10 |
(6) Transposition:
the 2 xtb buffer required for transposition was first prepared:
TABLE 12
Regent | Volume for 1ml |
1M Tris- |
20μl |
1M MgCl2 | 10μl |
10%NP40 | 10μl |
100%DMF | 200μl |
RSB-T-RI | 500 |
Nuclease-free Water | 260μl |
Then preparing a cell transposition reaction system:
watch 13
| Volume | |
2×TB | 25μl | |
Cells in RSB-T-RI | 10μl | |
PIC | 0.2μl | |
RI | 1μl | |
Tn5-1 | 5μl | |
Nuclease-free Water | 8.8μl | |
Total of | 50μl |
After a cell transposition system is configured in a PCR tube, the PCR tube is blown and beaten for 6 times by a gun head, and is placed in a constant temperature oscillator for transposition reaction at 37 ℃ for 30min. After the transposition is finished, collecting the transposition system in a 1.5ml low adsorption tube, washing the PCR tube once with precooled RSB-T-RI with the same volume, collecting the washed solution in the 1.5ml tube, and centrifuging for 5min at the temperature of 4 ℃ and 1000 g. The supernatant was removed and the cell pellet washed with 0.5ml of pre-cooled RSB-T-RI and centrifuged again at 1000g for 5min at 4 ℃. The supernatant was removed and the cells were resuspended with 60. Mu.l of pre-cooled RSB-T-RI.
(7) Reverse transcription:
first, a reverse transcription system is configured (taking 6 parts of reverse transcription reaction as an example):
TABLE 14
Note: 50% of PEG6000 is obtained by mixing PEG6000 with equal mass and water,
and (3) putting the PEG6000 with equal mass into a 65 ℃ water bath kettle, heating for 5min, and then cooling to room temperature.
Mu.l of the above reverse transcription mixture was added to 60. Mu.l of the post-transposition cells resuspended in RSB-T-RI in step (6). The mixture was blown evenly and divided into 6 PCR tubes, 50. Mu.l each. Then, in the PCR apparatus, the following procedure was followed:
watch 15
After the reverse transcription reaction is finished, collecting the reverse transcription product in a 1.5ml low adsorption tube, washing the PCR tube once with equal volume of RSB-T-RI, collecting the washed solution in the 1.5ml tube, and centrifuging for 5min at the temperature of 4 ℃ and 1000 g. The supernatant was removed and the cell pellet washed with 0.5ml of pre-cooled RSB-T-RI and centrifuged again at 1000g for 5min at 4 ℃. The supernatant was removed and the cells were resuspended with 1056. Mu.l of pre-cooled RSB-T-RI.
(8) Linkage of Barcode:
1. ligation of the first round of barcode:
first the hybridization buffer for the first round of barcode ligation was prepared as follows:
TABLE 16
Hybridization buffer | Volume(μl) |
NFw | 2761.9 |
T4 ligation buffer | 576 |
RI | 57.6 |
10%BSA | 57.6 |
In total | 3456 |
3168. Mu.l of the above hybridization buffer was added to 1056. Mu.l of the RSB-T-RI resuspended cells from step (7), passed through a 40 μm filter, then 100. Mu.l of T4 DNA ligase was added, and the mixture was inverted and mixed. Then, the Round1 96-well plate prepared in step (2) was added in a volume of 40. Mu.l per well, respectively. The plate was placed in a constant temperature shaker at 37 ℃ for 30min 300rpm.
After the reaction was completed, the Blocking oligo 1 mixture (see Table below) was prepared, and 10. Mu.l of the Blocking oligo 1 mixture was added to each well of Round 1-well plate, which was then placed in a constant temperature shaker at 37 ℃ for 300rpm 30min.
TABLE 17
2. Ligation of the second round of barcode:
the first Round of barcode ligation products in the 96-well plate were collected in 15ml tubes, 100. Mu.l of T4 DNA ligase was added, and the mixture was pipetted uniformly and then added to the Round2 96-well plate prepared in step (2) at a volume of 55. Mu.l per well, respectively. The plate was placed in a constant temperature shaker at 37 ℃ 300rpm 30min.
After the reaction, the Blocking oligo 2 mixture (Table below) was prepared, and 10. Mu.l of the Blocking oligo 2 mixture was added to each well of Round 2-well plate, followed by placing in a constant temperature shaker at 37 ℃ and 300rpm 30min.
Watch 18
|
Volume(μl) |
Round_2_blocking | 304 |
T4 ligation buffer | 211 |
NFw | 637 |
In total | 1152 |
3. Attachment of a third round of barcode:
the second Round of barcode ligation products in the 96-well plate were collected in 15ml tubes, 100. Mu.l of T4 DNA ligase was added thereto, and the mixture was pipetted uniformly and then added to the Round3 96-well plate prepared in step (2) in a volume of 70. Mu.l per well, respectively. The plate was placed in a constant temperature shaker at 37 ℃ 300rpm 30min.
After the reaction was completed, the Blocking oligo 3 mixture (see Table below) was prepared, and 10. Mu.l of the Blocking oligo 3 mixture was added to each well of Round 3-well plate, which was then placed in a constant temperature shaker at 37 ℃ for 300rpm 30min.
Watch 19
|
Volume(μl) |
Round_3_blocking | 265 |
Triton X-100 | 11.5 |
NFw | 875.5 |
Total of | 1152 |
4. The third round of barcode ligation product in a 96-well plate was collected in a 15ml tube, filtered through a 40 μm filter, centrifuged at 4 ℃ at 1000g for 5min, the supernatant was removed, the cells were resuspended in 1ml of RSB-T-RI, and again filtered through a 40 μm filter, centrifuged at 4 ℃ at 1000g for 5min, the pellet was collected, the cells were resuspended in 100 μ l of RSB-T-RI, and after pipetting well, 5 μ l was placed in a PCR tube and 5 μ l of 0.2% trawl was added.
(9) Isolation of ATAC library and RNA library:
1. cell de-crosslinking:
first, a 2x RCB that is uncrosslinked is configured:
watch 20
2x RCB | Volume(μl) |
1M Tris 8.0 | 100 |
5M NaCl | 20 |
10%SDS | 40 |
NFw | 840 |
Total of | 1000 |
Mu.l of the library was taken and 25. Mu.l of 2XRCB, 2. Mu.l of proteinase K, 1. Mu.l of RI were added to the library at 55 ℃ for 1h in a PCR instrument and hot lid 65 ℃.
Note: this step is followed by a stoppable point and the library after de-crosslinking can be stored at-20 ℃.
2. The above-mentioned uncrosslinked product was added to 2.5. Mu.l of 100mM PMSF, and incubated at room temperature for 10min while preparing the following solutions:
TABLE 21
1x B&W-T | Volume(μl) |
1M Tris 8.0 | 25 |
5M NaCl | 1000 |
0.5M |
5 |
10 |
25 |
NFw | 3945 |
Total of | 5000 |
TABLE 22
TABLE 23
2xB&W | Volume(μl) |
1M Tris 8.0 | 50 |
5M NaCl | 2000 |
0.5M |
10 |
NFw | 2940 |
Total of | 5000 |
Watch 24
1x B&W-T/RI | Volume/μl |
1x B&W-T | 600 |
RI | 6 |
TABLE 25
2x B&W/RI | Volume/ |
2x B&W | |
50 | |
|
1 |
Watch 26
1x STE/RI | Volume/μl |
STE | 300 |
|
1 |
3. And (3) taking 10 mu l of streptavidin magnetic beads, placing the streptavidin magnetic beads on a magnetic frame, removing the supernatant, then washing the streptavidin magnetic beads for three times by using 100 mu l of 1xB and W-T/RI, removing the supernatant, finally re-suspending the streptavidin magnetic beads by using 50 mu l of 2xB and W/RI, adding the re-suspended magnetic beads into the de-crosslinking product, uniformly blowing the solution by using a gun head, placing the solution in a 3D mixing rotator, and reacting for 1 hour at room temperature.
4. And (3) placing the PCR tube after the reaction is finished on a magnetic frame, and collecting the supernatant (ATAC library to be constructed) after the magnetic beads are adsorbed. The remaining beads were washed three times with 100. Mu.l of 1xB &W-T/RI, the supernatant removed, and the beads (RNA to be library) were then resuspended with 100. Mu.l of 1x STE/RI.
(10) Building a library of ATAC:
1. and purifying the ATAC library to be constructed by using a NEB DNA purification column, and eluting twice by using 11 mu l of EB and 12 mu l of EB respectively during elution to finally obtain 22.5 mu l of ATAC library to be constructed purified products.
2. The following PCR system was configured to perform amplification of the ATAC library:
watch 27
Regent | Volume(μl) |
NEBNext |
25 |
N5 | 1.25 |
S7 | 1.25 |
ATAC product | 22.5 |
Total of | 50 |
The PCR system was as follows:
watch 28
DNA magnetic bead purification PCR product:
add 20. Mu.l (0.4X) DNA magnetic beads into 50. Mu.l PCR system, blow and beat evenly with the gun, stand 5min at room temperature, place PCR tube on magnetic frame, take supernatant after magnetic beads are adsorbed, transfer to new PCR tube. Adding 40 μ l (1.2X) DNA magnetic beads again, blowing with a gun, standing at room temperature for 5min, removing supernatant after magnetic beads are adsorbed, washing magnetic beads with 80% ethanol twice, removing ethanol, and drying at room temperature for 3min. And finally, adding 20 mu l of NFw, blowing and beating uniformly, placing on a PCR plate, standing at room temperature for 5min, then placing on a magnetic frame, absorbing the supernatant in a new PCR tube after the magnetic beads are adsorbed, and obtaining a product, namely the ATAC library to be detected.
(11) RNA library construction:
template switching of RNA libraries:
taking the magnetic beads resuspended in 1 × STE/RI in the step (9), placing the magnetic beads on a magnetic frame, removing supernatant after the magnetic beads are adsorbed, and resuspending the magnetic beads by using a mixture converted by a template, wherein the configuration is as follows:
watch 29
Template switch mix | Volume(μl) |
50%PEG 6000 | 12.5 |
5x |
10 |
Ficoll PM-400(20%) | 10 |
10mM dNTPs,each | 5 |
|
5 |
|
5 |
Maxima RT RNase H Minus | 2.5 |
In |
50 |
Placing the resuspended magnetic beads in a 3D mixing and rotating instrument, incubating for 30min at room temperature, transferring to a constant temperature shaking instrument at 42 ℃ for reaction for 90min, and when reacting in the constant temperature shaking instrument, resuspending and uniformly mixing the magnetic beads by a gun every 30min.
2.cDNA Pre-amplification
The cDNA template conversion product was placed on a magnetic stand, the supernatant was removed after the beads were adsorbed, the beads were washed twice with 100. Mu.l of 1XSTE/RI, and the cDNA amplification mix in the following table resuspended the beads:
watch 30
cDNA PCR mix | Volume(μl) |
2× |
25 |
cDNA AMP F(20μM) | 1.25 |
cDNA AMP R(20μM) | 1.25 |
NFw | 22.5 |
In |
50 |
Then placed on a PCR instrument and the following reactions were performed:
watch 31
Purification of cDNA Pre-amplification product
Adding 40 (0.8 x) DNA magnetic beads into 50 mul cDNA pre-amplification product, blowing uniformly by a gun, standing for 5min at room temperature, placing a PCR tube on a magnetic frame, removing supernatant after the magnetic beads are adsorbed, cleaning the magnetic beads twice by 80% ethanol, removing the ethanol, and drying for 3min at room temperature. And finally, adding 20 mu l of NFw, blowing uniformly, placing on a PCR plate, placing for 5min at room temperature, then placing on a magnetic frame, absorbing the supernatant in a new PCR tube after magnetic beads are adsorbed, and obtaining a product, namely a cDNA pre-amplification purification product.
4.cDNA disruption to Bank and amplification
50ng of the cDNA pre-amplification purified product is taken for Tn5-2 interruption, and the system is as follows:
watch 32
| Volume | |
5×TTBL | 10μl | |
Tn5-2 | 5μl | |
cDNA | 50ng | |
NFw | 35-cDNAμl | |
In total | 50μl |
And (3) placing the reaction system in a constant temperature oscillator at 55 ℃ for 10min, after the reaction is finished, purifying the product after transposition by using a DNA purification column of NEB, and eluting twice by using 11 mu l of EB and 12 mu l of EB respectively during elution to finally obtain 22.5 mu l of Tag-cDNA purified product.
After purifying the product of the obtained Tag-cDNA, configuring an amplification reaction system as follows:
watch 33
Regent | Volume(μl) |
2× |
25 |
N5(20μM) | 1.25 |
S7(20μM) | 1.25 |
Tag-cDNA | 22.5 |
Total of | 50 |
The following amplification procedure was then performed:
watch 34
Purification of RNA library:
adding 35 (0.7 x) DNA magnetic beads into 50 mul of Tag-cDNA amplification products, blowing uniformly by using a gun, standing at room temperature for 5min, placing a PCR tube on a magnetic frame, removing supernatant after the magnetic beads are adsorbed, cleaning the magnetic beads twice by using 80% ethanol, removing the ethanol, and drying at room temperature for 3min. And finally, adding 20 mu l of NFw, blowing uniformly, placing on a PCR plate, placing for 5min at room temperature, then placing on a magnetic frame, absorbing the supernatant in a new PCR tube after magnetic beads are adsorbed, and obtaining a product, namely the RNA library to be detected.
(12) Quality testing and sequencing of the library:
1. and (3) using the Qubit to measure the concentration of dsDNA of the library to be tested of ATAC and RNA, wherein the concentration of the library is required to be more than 10ng/ul.
2. Taking 10ng of ATAC and RNA to be detected library, running 4200 D1000 gel, wherein the distribution of ATAC should be 300-700 bp, the nucleosome peak type is provided, and the first main peak is about 340 bp; the distribution of RNA should be 300-700 bp, and the main peak is around 450 bp.
Example 2
Example 1 the results of the library constructed as compared to the 10x platform or SHARE-seq are as follows:
1. the measured uniq fragments (only valid fragments) of each cell were at the same level in the ATAC library (see FIG. 4);
2: the difference is caused by the fact that FRiTSS (TSS enrichment ratio) is relatively low in ATAC library, and the difference is caused by different samples, and the FRiTSS of the 3T3 cell line of xseq is not greatly different from the FRiTSS of the 3T3 cell line of SHARE (see figure 5);
3: the number of genes detected in each cell was not very different in the RNA library compared to the SHARE-seq (see FIG. 6);
4: in the RNA library, the number of UMI (unique transcripts) detected per cell was not very different compared to the SHARE-seq (see FIG. 7).
The above results show that the ATAC library and RNA library constructed by the present invention have comparable quality to the 10X platform, SHARE-seq.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.
Claims (10)
1. A Tn5 transposase complex is characterized by comprising Tn5 transposase and a first assembly sequence, wherein the first assembly sequence comprises a sequence with a nucleotide sequence shown as SEQ ID NO. 1-2.
2. The Tn5 transposase complex of claim 1, wherein the Tn5 transposase complex further comprises a second assembly sequence;
the second assembly sequence comprises a sequence with a nucleotide sequence shown as SEQ ID NO.1 and a sequence shown as SEQ ID NO. 3.
3. The Tn5 transposase complex as claimed in claim 1 or 2, wherein the sequence shown in SEQ ID No.1 has a phosphorylation modification at the 5 'end and a dideoxy modification at the 3' end; the 5' end of the sequence shown in SEQ ID NO.3 has phosphorylation modification.
4. A method for constructing a single-cell multiomic library, which is characterized by comprising the following steps:
(1) Fixing the cells, and performing permeabilization treatment;
(2) Transposing the cells treated in step (1) with the transposase complex of claim 2 to obtain transposed cells; the transposed cells contain labeled chromatin opening regions and unlabeled mRNA.
(3) Reverse transcription of mRNA in the cells obtained in step (2) by using a reverse transcription primer; the reverse transcription primer sequence is shown as SEQ ID NO.295;
(4) Sequentially carrying out first round, second round and third round Barcode labeling on the reverse transcription product in the step (3) by using primers;
(5) Collecting the cells marked in the step (4), performing crosslinking release, separating cDNA (complementary deoxyribonucleic acid) and chromatin by using streptavidin magnetic beads, and respectively building libraries to obtain an RNA (ribonucleic acid) library and an ATAC (atom transfer assay) library;
in the RNA library construction process, the Tn5 transposase complex of claim 1 is used to fragment cDNA.
5. The method according to claim 4, wherein in the step (1), the fixing agent is formaldehyde, and the agent for permeabilization is NP40.
6. The method according to claim 4, wherein in the step (2), the reverse transcription primer is modified with biotin.
7. The method for constructing a plasmid according to claim 4, wherein in the step (4), the first Round of Barcode-labeled primers comprise Round1linker and Round1 Barcode; the second Round of Barcode labeled primers comprise Round2 linker and Round2 Barcode; the third Round of Barcode labeled primers comprise Round3linker and Round3 Barcode;
the Round1 Barcode sequentially comprises a 5' phosphorylation marker, a sequence complementary to a part of bases of Round2 linker marked by the second Round Barcode, a first Round Barcode and a sequence complementary to a part of bases of Round1linker marked by the first Round Barcode from 5' to 3 ';
the Round2 Barcode comprises a 5' phosphorylation label, a sequence which is complementary with a Round3linker part base labeled by the third Round Barcode, a second Round Barcode and a sequence which is complementary with a Round2 linker part base labeled by the second Round Barcode from 5' to 3 ';
the Round 3Barcode sequentially comprises a sequence complementary to an upstream primer amplified from the cDNA library, a UMI sequence, a third Round of Barcode and a sequence complementary to a part of bases of Round3linker labeled with the third Round of Barcode from 5 'to 3'.
8. The construction method according to claim 7, wherein the nucleotide sequence of Round1linker is shown in SEQ ID NO.289, and the nucleotide sequence of Round1 barcode is shown in SEQ ID NO. 1-96;
the nucleotide sequence of the Round2 linker is shown as SEQ ID NO.290, and the nucleotide sequence of the Round2 barcode is shown as SEQ ID NO. 97-192;
the nucleotide sequence of Round3linker is shown in SEQ ID NO.291, and the nucleotide sequence of Round 3barcode is shown in SEQ ID NO. 193-288.
9. The building method according to claim 4, wherein the step (5) specifically comprises:
(a) Collecting the cells marked in the step (4), and performing crosslinking; separating the uncrosslinked product by streptavidin magnetic beads to obtain cDNA and chromatin open regions;
(b) Purifying the chromatin open region obtained by separation in the step (a), and performing PCR amplification, enrichment and purification to obtain an ATAC library;
(c) Taking cDNA obtained by separation in the step (a), pre-amplifying, purifying, adding a transposase compound of claim 1 to obtain a Tag-cDNA fragmentation product; and carrying out PCR amplification, enrichment and purification on the purified product of the Tag-cDNA to obtain an RNA library.
10. The library obtained by the construction method according to any one of claims 4 to 9, which includes an RNA library and an ATAC library.
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