High throughout sequencing of paired VH and VL transcripts from B cells secreting antigen-specific antibodies
View Patent ↗Methods for determining the VH:VL antibody repertoire from cells, such as antigen-specific B cells, in a high throughput manner. In some aspects, methods are provided for the capture of mRNA transcripts from single B cells that secreted antigen-specific antibodies followed by sequencing of the corresponding cDNAs encoding antibody VH and VL sequences. Libraries of antibodies produced by such methods are also provided.
1. A method for identifying the sequence of two or more transcripts from a plurality of single cells comprising:
a) incubating isolated single cells and capture agents in individual compartments, wherein the capture agents bind to both mRNA and antibodies;
b) lysing the isolated cells;
c) contacting the capture agents, along with bound mRNA and antibodies, with affinity agents that bind to antibodies to form complexes;
d) purifying the complexes;
e) performing reverse transcription and PCR amplification on individual complexes to generate linked amplification products comprising two or more cDNAs from said cell; and
e) sequencing amplification products to identify antibody sequences from a single cell.
2. The method of claim 1 , wherein the plurality of single cells comprise a plurality of individual B cells.
3. The method of claim 2 , wherein the individual B cells are primary B-cells from a human subject.
4. The method of claim 1 , wherein the capture agents are beads.
5. The method of claim 1 , wherein the capture agents comprise oligonucleotides which hybridize mRNA.
6. The method of claim 1 , wherein said sequencing comprises sequencing antibody VH and VL sequences for an antibody that is bound to an antigen of interest.
7. The method of claim 6 , wherein the capture agents comprise the antigen of interest.
8. The method of claim 7 , wherein the capture agents are beads that comprise the antigen of interest and a mRNA binding moiety.
9. The method of claim 1 , wherein the affinity agents are beads.
10. The method of claim 9 , wherein the affinity agents comprise polystyrene beads.
11. The method of claim 9 , wherein the affinity agents are magnetic beads.
12. The method of claim 1 , wherein the affinity agents have density (mass per unit volume) that is less than the capture agents.
13. The method of claim 1 , wherein the affinity agents bind to an antibody constant region.
14. The method of claim 1 , wherein the individual compartments are wells in a gel or microtiter plate.
15. The method of claim 1 , wherein the compartments are sealed with a permeable membrane prior to lysing the individual cells.
16. The method of claim 1 , wherein lysing the isolated cells comprises the use of a mild detergent solution.
17. The method of claim 1 , comprising identifying antibody sequences from at least 10,000 individual cells.
18. The method of claim 1 , wherein purifying the complexes comprises separating complexes by magnetic properties or by density.
19. The method of claim 1 , wherein step (e) comprises performing emulsion-reverse transcription and PCR amplification on individual complexes.