High-throughput polynucleotide library sequencing and transcriptome analysis

A polynucleotide and target polynucleotide technology, which is applied in the field of high-throughput polynucleotide library sequencing and transcriptome analysis, and can solve problems such as limited throughput and inability to capture full-length immune receptor sequences.

Pending Publication Date: 2020-05-12
ABVITRO LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing tools for single-cell transcriptome sequencing are limited in...

Method used

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  • High-throughput polynucleotide library sequencing and transcriptome analysis
  • High-throughput polynucleotide library sequencing and transcriptome analysis
  • High-throughput polynucleotide library sequencing and transcriptome analysis

Examples

Experimental program
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Embodiment 1

[0672] Example 1- Barcoding transcripts in emulsions for single-cell polynucleotide sequencing

[0673] A. Cell Preparation

[0674] Single cell suspensions for single cell polynucleotide sequencing were obtained from total peripheral blood mononuclear cells (PBMC). Withdraw approximately 50 mL of blood into a Vacutainer CPT cell preparation tube with sodium heparin (BD), centrifuge at 1800 x g for 20 min, wash in cell preparation buffer (1x PBS supplemented with 2% fetal bovine serum and 2 mM EDTA) Twice, platelets were removed using a spin at 200 xg, and the resulting PBMCs were stored cryopreserved at -80°C in RPMI-1640 medium (Life Technologies) + 20% fetal bovine serum + 10% DMSO until needed. Prior to emulsion production, PBMCs were thawed and washed twice by centrifugation (200 xg for 10 min) in cell buffer 1 x Dulbecco's phosphate buffered saline (PBS). Cells were then diluted in cell buffer to 3.5 x 10 6 cell concentration per cell / mL. The suspension was then pipe...

Embodiment 2

[0693] Example 2- Barcoding Transcripts in Emulsions Using Target-Specific Primers for Single-Cell Polynucleotide Sequencing

[0694] A. Cell Preparation

[0695] Draw 50 mL of blood into a Vacutainer CPT cell preparation tube with sodium heparin (BD), centrifuge at 1800 x g for 20 minutes, wash twice in cell preparation buffer (1x PBS supplemented with 2% fetal bovine serum and 2 mM EDTA), Platelets were removed using a spin at 200 xg, and the resulting PBMCs were stored cryopreserved at -80°C in RPMI-1640 medium (Life Technologies) + 20% fetal calf serum + 10% DMSO until needed. Prior to emulsion generation, PBMCs were thawed, washed twice in cell preparation buffer and counted. B cells were isolated using a negative selection based human B cell enrichment kit (StemCell Technologies). Pass cells through a 20 micron cell strainer and dilute to 6.2E+06 cells / ml (3 million B cell experiments) or 3.1E+06 cells / ml (PGT donor and ovarian tumor experiments) in cell preparation ...

Embodiment 3

[0702] Example 3- Barcoding of Target Sequences in Emulsions and Transcriptome Transcripts for Single-Cell Polynucleotide Sequencing method

[0703] A. Cell Preparation

[0704]Cryopreserved PBMC suspensions were thawed quickly and added to 10 volumes of RPMI+10% FBS at room temperature. Cells were pelleted by centrifugation at 350 x g for 8 minutes and resuspended at 2 x 10^6 cells / mL in RPMI + 10% FBS. Allow the PBMCs to rest in the tissue culture incubator for approximately 16 hours.

[0705] Resting PBMCs were co-cultured with autologous antigen-presenting cells at a ratio of approximately 10:1 PBMC:APC. In this case, APCs were autologous monocyte-derived dendritic cells that had been exposed to irradiated HSV-infected HeLa cells. Co-cultures were incubated for 5 hours. Without intending to be limited to the methods described herein, it is contemplated that an incubation time of about 5 hours is sufficient to allow antigen-specific cells to express new mRNA in resp...

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Abstract

Provided herein are methods for target gene sequencing and single cell barcoding in conjunction with analysis of gene expression in single cells. In some embodiments, the target gene is an immune molecule, such as an antibody or TCR. In some embodiments, the methods can be used to carry out transcriptome sequencing, e.g., RNA sequencing, to capture transcriptome of single cells paired with full receptor immune receptor sequences such that information about the immune repertoire and transcriptome of a cell can be determined. Also provided are polynucleotide libraries for use in carrying out transcriptome analysis and immune molecule, e.g., antibody or TCR, sequencing.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to U.S. Provisional Application No. 62 / 511,949, filed May 26, 2017, entitled "HIGH-THROUGHPUT POLYNUCLEOTIDE LIBRARY SEQUENCING AND TRANSCRIPTOMEANALYSIS," and will Its content is incorporated by reference in its entirety. [0003] Incorporated by reference into the sequence listing [0004] This application is filed with a Sequence Listing in electronic format. The Sequence Listing is provided as a file titled 735042011740SeqList.txt created on May 25, 2018, which is 444,416 bytes in size. The information in the sequence listing in electronic format is incorporated by reference in its entirety. technical field [0005] The present disclosure relates to methods for target gene sequencing and single cell barcoding combined with analysis of gene expression in single cells. In some embodiments, the target gene is an immune molecule, such as an antibody or TCR. In some embodiments, th...

Claims

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Application Information

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IPC IPC(8): C12Q1/6855
CPCC12Q1/6855C12Q2525/155C12Q2525/179C12Q2525/191C12Q2563/159C12Q2563/179C40B40/06
Inventor S·J·戈德弗莱斯A·W·布里格斯R·查里Y·蒋R·豪斯F·维尼奥特
Owner ABVITRO LLC
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