37 results about "CDNA library" patented technology

The invention provides a frozen section method for space transcriptomics analysis of mesentery and regenerative intestinal tract of stichopus japonicus, and belongs to the technical field of biotechnology. The method comprises the steps of sample treatment and embedding, pre-cooling by a freezing slicer, freezing and slicing, and flaking. According to the frozen section method for space transcriptome analysis of the mesentery and the regenerative intestinal tract of the stichopus japonicus, provided by the invention, a fresh material is directly embedded, and a plurality of thin tissue blocks with the same size are integrated into a standard tissue block of 1cm, so that the high coverage rate of a tissue cross section in a space transcriptome chip capture area is ensured; rNA degradation is effectively prevented, and finally, the cDNA library is successfully constructed and is used for subsequent analysis of a space transcriptome. The technical scheme has the characteristics of simplicity and convenience in operation and stable effect, and is particularly suitable for space transcriptomics frozen section preparation of thin film type tissues.

The invention discloses a sequencing method of an immune repertoire, which comprises the following steps: 1) constructing a cDNA library of a sample, the cDNA in the cDNA library being connected with the position information of the cDNA on the sample; 2) enriching cDNA of TCR and / or BCR genes in the cDNA library in a targeted manner to obtain an immune repertoire of the sample; and (3) sequencing the immune repertoire. According to the method disclosed by the invention, targeted enrichment and high-throughput sequencing of TCR and / or BCR sequences are innovatively realized, and gene expression information and spatial position information of each cell in a space are detected at the same time.

A high-throughput screening system and method for small molecule-protein interactions includes first immobilizing selected small molecules on a solid matrix. Immobilization is preferably performed via photoaffinity labeling, where a linker is used to bind the small molecule to the matrix. cDNA libraries are constructed based on various human tissues. Tissue mRNA is transcribed into cDNA and transferred to a phage genome to construct a phage display library, which is then mixed with small molecules conjugated to microspheres and incubated. The incubated mixture is washed to remove unbound phages, including non-specific and weakly bound phages. Bound phages are amplified and used for the next round of biopanning. The biopanning cycle is repeated, and then PCR is performed on each plaque obtained from the final round of biopanning. The PCR products from each small molecule experiment are barcoded, merged, and then subjected to next-generation sequencing.

Systems and methods for long-read sequencing of immune receptor are disclosed. A method in accordance with the present disclosure comprises generating a 3'-barcoded cDNA library comprising an immune receptor target sequence, wherein the immune receptor target sequence comprises a variable region and a barcoded region; amplifying the immune receptor target sequence; linking the variable region of the immune receptor target sequence to a probe to generate a labeled immune receptor target sequence; capturing the labeled immune receptor target sequence; amplifying the captured immune receptor target sequence; passing the captured immune receptor target sequence through a nanopore sequencing device; and generating, by way of the nanopore sequencing device, a full-length immune receptor target sequence.

The application provides a prostate cancer biochemical recurrence prognosis risk prediction model based on fatty acid metabolism and cancer cell stemness genes and a construction method thereof, wherein the construction method comprises the following steps: S1: data collection: obtaining prostate cancer sample transcriptome data with biochemical recurrence information from a database, and dividing the data into a model training set and a model test set; S2: stemness score analysis; S3: fatty acid metabolism score analysis; S4: based on the analysis results of S2 and S3, identifying a co-expression gene module related to fatty acid metabolism and stemness characteristics in prostate cancer by a co-expression similarity algorithm and a hierarchical clustering algorithm, and obtaining a fatty acid metabolism and stemness-related gene set; S5: constructing a prostate cancer BCR prognosis risk prediction model; S6: constructing a nomogram model; S7: extracting RNA of a to-be-tested sample, constructing a cDNA library, quantifying the expression of the above genes, and calculating the prognosis risk level of prostate cancer through the expression level.

The invention discloses a method for drawing a human full-coverage protein interaction network based on digital PCR next-generation sequencing. After the advanced pedestrian 293T cell line obtains mRNA, a modified random primer or an oligo-dT primer is adopted to obtain a cDNA library; carrying out homogenization treatment on the human cDNA library; the method comprises the following steps: transforming plasmids of a BACTH bacteria double-hybrid system to obtain transformed plasmids; carrying out homologous recombination on the transformed plasmids of the sample library and the double-impurity system, and introducing into escherichia coli for screening to obtain positive PPI clones of the library with interaction; and carrying out digital PCR-based next-generation sequencing on the screened clones to draw the human full-coverage protein interaction network. The method is suitable for performing high-throughput screening after thousands of positive PPI combinatorial clones are obtained by'library-to-library 'bacteria or yeast double hybrids, identifying the same cell bar code cDNA combinatorial pairs, and realizing PPI network identification in different species, among species and in hybridization technology system extensive scenes.

Synthetic polynucleotide libraries may include a plurality of polynucleotides. The polynucleotides may comprise DNA and may be configured to hybridize with one or more regions of target nucleic acids. The target nucleic acids may comprise a cDNA library. The cDNA library may comprise at least one exon-exon boundary between a first exon and a second exon.

Herein provided is a novel method, nanoPARE (parallel analysis of RNA ends from low-input RNA), for generating multiple sequencing libraries from a single full-length cDNA library, specifically an RNA 5′ end sequencing library and / or a gene body cDNA library, and method for analyzing RNA 5′ ends, and optionally associated software developed therefor. Further provided is a cDNA library generated therefor.

The application discloses a high-throughput general sample protein interaction screening method. The method comprises the following steps: firstly, constructing a general sample open reading frame library, extracting mRNA of a target sample, and then obtaining a cDNA library by using a modified random primer or an oligo-dT primer; then, uniformly processing the general sample cDNA library; modifying plasmids of a double-hybrid system to obtain modified plasmids; homologously recombining the general sample library and the modified plasmids of the double-hybrid system, and introducing them into organisms to screen and obtain organisms with an interaction library; identifying single-cell PPI pairs of the screened organisms, and constructing a general sample protein interaction network. The application is suitable for screening and identifying the same Barcode cDNA combination pairs by using single-cell sequencing after obtaining tens of thousands of positive PPI combination clones by means of a "library-library" bacterial or yeast double-hybrid system, and realizing PPI network identification in different intra-species, inter-species and hybridization technology systems.

The invention belongs to the technical field of biology, and particularly discloses a TCR / BCR space transcriptome sequencing method. The method is characterized in that a unique space transcriptome chip is adopted, and a capture probe of the chip sequentially comprises a universal sequence 1, a space address code, a unique molecular tag, a universal sequence 2 and a Poly dT sequence; after a cDNA library is constructed by using the chip, cyclizing the cDNA library, so that a spatial address code is physically adjacent to a variable region; and then taking the cyclized DNA as a template, and carrying out multiple PCR amplification by using a primer complementary with the universal sequence 2 and a specific primer aiming at the constant region, so as to enrich a fragment simultaneously containing a complete variable region sequence and a space address code. Through cooperation of chip design and cyclization enrichment, a variable region primer pool is not needed, and the space variable region sequence of the immune receptor can be captured in an unbiased and efficient manner.

The application discloses a method for detecting viruses and virus replication in media based on Small-RNA high-throughput sequencing. The application belongs to the technical field of virus detection, and the method for identifying virus types in a to-be-detected sample by Small-RNA sequencing developed by the application comprises the following steps: 1) extracting total RNA from a virus-infected to-be-detected sample, constructing a cDNA library, and separating small RNA sequence fragments from the cDNA library; 2) downloading virus sequences and classification databases from GenBank, and comparing the small RNA sequence fragments with virus sequence files; 3) comparing a contigs3 file with a virus nucleic acid database, selecting sequences with the minimum e value in the comparison results in the contigs3 file, calculating the coverage of the contigs3 sequences in corresponding virus genomes, distinguishing virus information with a coverage threshold, and deducing the virus source of the small RNA sequence.

The invention belongs to the technical field of gene sequencing, and particularly relates to a library building kit and probe for detecting blood tumor related fusion genes. According to the library building kit, only a trace amount of total RNA is needed to be used for cDNA synthesis, the initial quantity of a template can be as low as 10 ng, so that a cDNA library is built, then the library is subjected to hybrid capture and enrichment by using an autonomously designed probe hematologic tumor fusion gene Panel (RA Panel), the time consumption is short, the efficiency is high, and simultaneous building and on-machine sequencing of 384 libraries can be realized. According to the rapid hybridization system of the library building kit, the shortest hybridization time can reach 30 min, the size of a finally obtained library fragment is about 200-400 bp, and the rapid hybridization system can be compatible with various Illumina sequencing platforms for sequencing. According to the invention, through a simple and easy-to-use solution of NGS sequencing while synthesis, rapid and accurate typing of fusion genes which may affect blood tumor diagnosis is realized.

The application discloses a method for drawing a human full-coverage protein interaction network based on digital PCR and second-generation sequencing. First, mRNA is obtained from a human 293T cell line, and then a cDNA library is obtained by using a modified random primer or an oligo-dT primer; then, the human cDNA library is uniformly processed; the plasmid of a BACTH bacterial double-hybrid system is modified to obtain a modified plasmid; the sample library and the modified plasmid of the double-hybrid system are homologously recombined and introduced into E. coli to screen positive PPI clones with interaction; and the screened clones are subjected to second-generation sequencing based on digital PCR to draw a human full-coverage protein interaction network. The application is suitable for high-throughput screening after obtaining tens of thousands of positive PPI combination clones by using a "library vs. library" bacterial or yeast double-hybrid system, and is suitable for identifying the same cell barcode cDNA combination to realize PPI network identification in different intra-species, inter-species and hybridization technology systems.

A method for preparing interaction plasmid library and a method for testing interactions between cDNA libraries are provided. The cDNA libraries could be inserted into the first multiple cloning site and the second multiple cloning site of the dual expression vector for testing the interactions between the different cDNA libraries.

Systems and methods for long-read sequencing of immune receptor are disclosed. A method in accordance with the present disclosure comprises generating a 3'-barcoded cDNA library comprising an immune receptor target sequence, wherein the immune receptor target sequence comprises a variable region and a barcoded region; amplifying the immune receptor target sequence; linking the variable region of the immune receptor target sequence to a probe to generate a labeled immune receptor target sequence; capturing the labeled immune receptor target sequence; amplifying the captured immune receptor target sequence; passing the captured immune receptor target sequence through a nanopore sequencing device; and generating, by way of the nanopore sequencing device, a full-length immune receptor target sequence.

The invention relates to an mRNA vaccine quality evaluation method based on a nanopore sequencing platform, which comprises the following steps: S1, preparation and sequencing of a long read length library: carrying out reverse transcription on an mRNA vaccine sample to prepare a cDNA library, and sequencing to obtain full-length cDNA sequencing data of the sample; s2, sequencing data acquisition and quality control: performing real-time sequencing, and then performing quality control to obtain clean reads data; s3, sequencing data comparison and analysis: comparing clean reads data to a design sequence, carrying out mRNA structural integrity analysis, and identifying sequence information, and S4, mRNA vaccine quality evaluation system construction and verification: establishing a multi-dimensional quality evaluation index system including sequence purity, integrity, fracture sites, sequence consistency and PolyA length. According to the method, the key quality attributes of the mRNA vaccine can be comprehensively measured, so that the production efficiency and reliability of the mRNA vaccine are improved.

The invention discloses an antibacterial peptide Hyastatin gene of Eriocheir sinensis as well as a recombinant mature peptide and application of the antibacterial peptide Hyastatin gene. A unigene sequence obtained by transcriptome sequencing is utilized, a 5'and 3 'terminal rapid amplification technology is utilized to amplify in an Eriocheir sinensis blood lymphocyte cDNA library to obtain an antibacterial peptide Hyststin gene full-length sequence, and the gene plays an important role in the innate immune defense aspect of Eriocheir sinensis. According to the present invention, the Eriocheir sinensis antibacterial peptide Hyastatin recombinant mature peptide is obtained by using the in vitro recombinant expression technology, the antibacterial peptide Hyastatin recombinant mature peptide has significant inhibition effect on the common aquaculture pathogenic bacteria aeromonas hydrophila, and the foundation can be laid for Eriocheir sinensis disease control, medical product development and feed additive application.

The present invention relates to the field of biotechnology and a method for creating a cDNA library. More specifically, the invention refers to a method of forming complementary DNA (cDNA) sequencing libraries from RNA in situ comprising the steps of: (a) fixating cells, immobilized on a solid surface; (b) performing an in situ reverse transcription (RT) inside cells, using RT primers comprising a PCR handle 1, and partially biotinylated dNTPs; (c) releasing single stranded cDNA from the cells using a release mix, wherein the release mix comprises an RNAse, such that the single stranded cDNA is released from intact cells; (d) collecting a supernatant comprising released cDNA into a single larger volume or in separate volumes; and (e) introducing an adapter molecule comprising a PCR handle 2 that will bind 3′ of the extended cDNA.

The invention provides a transcription factor CsNF-YA and application thereof in positive regulation of CsCBCAS gene expression of China-hemp, and belongs to the technical field of gene engineering. The invention provides a transcription factor CsNF-YA, and the nucleotide sequence of the transcription factor CsNF-YA is as shown in SEQ ID No.2. The transcription factor CsNF-YA is screened from a China-hemp yeast cDNA library, the transcription factor CsNF-YA can be specifically combined with a China-hemp CsCBCAS gene promoter, expression of the CsCBCAS gene can be remarkably promoted by overexpression of the transcription factor CsNF-YA, and the transcription factor CsNF-YA can be applied to synthesis genetic improvement of China-hemp CBC.

The invention discloses a method for drawing a mouse full-coverage protein interaction network through bacterial single cell transcriptome sequencing. The method comprises the following steps: firstly, extracting mRNA (messenger ribonucleic acid) of pan-tissue of a mouse, and then obtaining a cDNA (complementary deoxyribonucleic acid) library by adopting a modified random primer or an oligo-dT primer; carrying out homogenization treatment on the mouse protein library; the method comprises the following steps: transforming plasmids of a BACTH bacteria double-hybrid system to obtain transformed plasmids; carrying out homologous recombination on the transformed plasmids of the sample library and the double hybrid system, and introducing into DHM1 escherichia coli for screening to obtain positive PPI clone with a mutual composition library; and carrying out single-cell transcriptome sequencing on the screened clones based on random primers to construct the mouse protein interaction network. The method is used for carrying out high-throughput screening on mice after thousands of positive PPI combinatorial clones are obtained by'library-to-library 'bacteria or yeast double hybrids, and identifying the same Barcode cDNA combinatorial pairs to realize PPI network identification in different species, among species and in hybridization technology system extensive scenes.

The invention relates to the technical field of biology, in particular to a racoon ussuriensis dog skin hair follicle single cell transcriptome map and a construction method thereof. The construction method comprises the following steps: collecting a skin tissue sample in the middle of the back of a male racoon ussuriensis dog in a winter hair period, dissociating and preparing a single-cell suspension, constructing a cDNA library and sequencing; comparing original sequencing data to a reference genome, and quantifying high-throughput single cell transcriptome data; further quality control is carried out, low-quality cells and double cells are removed, and standardization processing is carried out on data; screening hypervariable genes, and performing dimensionality reduction and visualization on a gene expression profile to obtain marker genes of a cell population; and calculating the correlation between the to-be-identified cell expression profile and the reference data set, and identifying the cell type. Based on a single cell transcriptome sequencing technology, the cell heterogeneity problem which cannot be solved by tissue sample sequencing is solved, a new way is provided for exploring a hair follicle development heterogeneity mechanism, and a new direction is also provided for biological research of racoon dog hair follicles.

The present invention discloses a method for combining spatial transcriptomics and CRISPR screening in a same-slice unbiased manner and its application in gene function verification, belonging to the field of biotechnology. The steps of the present invention include: infecting target cells with a CRISPR screening library virus; sorting out positively infected target cells and culturing them for a period of time after knockout occurs; injecting the target cells obtained in the foregoing steps into an experimental animal, where the experimental animal is an animal with autoimmune deficiency; taking out the tissue infiltrated by the target cells and preparing frozen sections; transferring the frozen sections onto a spatial transcriptomics slide and loading it onto a machine to obtain a spatial transcriptomics cDNA library; constructing a general transcriptome library and a CRISPR sgRNA library using the spatial transcriptomics cDNA library; respectively sequencing and comparing gene sequences of the general transcriptome library and the CRISPR sgRNA library to obtain general transcriptome sequencing data and CRISPR sgRNA library sequencing data carrying spatial coordinates.

Disclosed herein are compositions and methods related to the fast and efficient generation of cDNA library from RNA. The method allows integration of the method in an automation adaptable system.

The present invention discloses a sc3T-seq method for accurately analyzing APA sites in single cells, which relates to the fields of molecular biology and bioinformatics. The method comprises incubation of oligo(dT)20 primers with streptavidin magnetic beads, addition of a small amount of starting sample and exogenous single-stranded DNA, synthesis of double-stranded cDNA, breakage of the double-stranded cDNA, removal of polyA, release of labeled cDNA, addition of biotin-labeled λ-DNA, addition of carboxyl magnetic beads, screening and removal of exogenous cDNA, end repair, addition of linkers, construction of a cDNA library, and removal of biotin-labeled λDNA with streptavidin magnetic beads to obtain a purified sample library. The present invention reduces the amount of starting sample, making the experimental technique for analyzing APA sites applicable to a small amount of samples or even single cells, and has a great effect on the accurate detection and analysis of cell-specific APAs.

The application discloses a sequencing analysis method for single-cell transcriptome based on ONT sequencing and relates to the technical field of gene sequencing analysis.The method comprises the following steps: preparing and constructing a cDNA library of a sample to be measured, sequencing, obtaining three generations of original data, performing quality control on the three generations of original data, obtaining three generations of effective data, performing functional annotation on a reference genome, using software ONT-SC-pipeline to perform data splitting, alignment and quantification, obtaining quantitative data, sequentially performing basic analysis, Marker gene enrichment analysis and advanced analysis on the quantitative data, wherein the basic analysis comprises top gene distribution analysis, hyper-variable gene analysis, clustering and grouping analysis, correlation analysis of each cell group, dimension reduction analysis and Marker gene identification; and the advanced analysis comprises cell annotation analysis, cell communication analysis, cell trajectory analysis, variable splicing analysis and fusion gene analysis.

This invention provides a method for targeted enrichment of target sequences in a spatial transcriptome cDNA library. The method includes: using a hybridization capture probe set to hybridize and extend a target nucleic acid sequence or its complementary sequence on the target sequence in the spatial transcriptome cDNA library to obtain an extension product; wherein the hybridization capture probe set includes two or more probes, and any two probes in the hybridization capture probe set do not overlap in the hybridization region of the target sequence; the extension direction of any probe in the hybridization capture probe set is from the target nucleic acid sequence or its complementary sequence to the spatial sequence or its complementary sequence; the extension product includes: the spatial sequence or its complementary sequence, and part or all of the target nucleic acid sequence or its complementary sequence; the extension product is isolated to obtain an enriched library. This method solves the problem in existing technologies that only unbiased sequencing can be performed on spatial transcriptome libraries, and targeted sequencing cannot be achieved, making it applicable to the field of spatial transcriptome library enrichment.

The present application provides a spatial transcriptome chip, a method for constructing a cDNA library, and a method for transcriptome sequencing analysis. The chip includes a substrate, on which multiple probe groups are fixed, each of which includes a first probe and a second probe; the first probe includes a sequencing primer segment, a barcode segment, a specific molecular tag, and a first capture structure for capturing the 3' end of mRNA, which are sequentially connected from the substrate upward; the second probe includes a sequencing primer segment, a barcode segment, a specific molecular tag, and a second capture structure for capturing the 5' end of mRNA, which are sequentially connected from the substrate upward; the first probe and the second probe in each probe group have the same barcode segment, and at least two of the multiple probe groups have different barcode segments.

The present invention discloses a method and a kit for whole transcriptome sequencing of a small amount of tumor cells from fresh samples. Starting from a sample lysed from more than 5000 tumor cells within 8-9 hours, the first strand of cDNA is obtained and then the second strand of cDNA is generated. While exponentially amplifying with the linker segment as the primer anchoring site, Illumina library linkers are added to both ends of the cDNA to obtain a high-quality cDNA library meeting the requirements of downstream analysis. It can amplify 10-20 ng of high-quality full-length double-stranded cDNA, with a reverse transcription and amplification library construction success rate of more than 95%. The cDNA library is fragmented by the transposase method; after testing, the lysis solution has good lysis effects on different tumor cells, can effectively exclude other interferences, ensure the integrity and concentration of RNA, and can ensure that all tumor cells are lysed while the RNA is not degraded; at the same time, the designed reverse transcription primer polyT is linked with biotin at the 5' segment, and through specific binding with streptavidin magnetic beads, the influence on subsequent data caused by genomic contamination can be effectively avoided.

The invention discloses a method for screening a melon cold-resistant gene based on a cDNA yeast library, and belongs to the field of gene engineering. The method comprises the following steps: sampling melon leaves subjected to low-temperature treatment, extracting total RNA (Ribonucleic Acid), carrying out reverse transcription to obtain a high-coverage-rate cDNA (Complementary Deoxyribonucleic Acid) library, connecting the cDNA library with a linker to form a three-frame library, connecting the cDNA library to a pYES-NTB carrier, electrically transforming the obtained cDNA library into an escherichia coli competent cell, carrying out cloning amplification, and extracting library plasmids; and transforming the library plasmid and the pYES2 vector into a yeast strain BY4741, screening at low temperature of-20 DEG C for 72 hours, collecting cold-resistant clones, and amplifying and sequencing to obtain the cold-resistant gene of the muskmelon. The constructed yeast system is utilized to efficiently identify potential candidate genes for improving the cold resistance of the muskmelon seedlings, and a foundation is laid for genetic engineering breeding of low-temperature stress resistance of muskmelons.

The application discloses a method for drawing a mouse full-coverage protein interaction network by bacterial single-cell transcriptome sequencing. The method comprises the following steps: extracting mRNA of a mouse general tissue, obtaining a cDNA library by using a modified random primer or an oligo-dT primer, uniformly processing a mouse protein library, modifying a plasmid of a BACTH bacterial double-hybrid system to obtain a modified plasmid, homologous recombining the sample library and the modified plasmid of the double-hybrid system, introducing into DHM1 E. coli to screen positive PPI clones with an interaction library, and performing single-cell transcriptome sequencing based on the random primer on the screened clones to construct a mouse protein interaction network. The application is used for high-throughput screening of tens of thousands of positive PPI combination clones obtained by a 'library versus library' bacterial or yeast double-hybrid method, and identification of the same Barcode cDNA combination to realize PPI network identification in different intra-species, inter-species and hybridization technology systems.