Single cell bar-coding for antibody discovery

A method comprising, in at least one vessel of a plurality of vessels, the at least one vessel comprising (i) an immune cell;

• (ii) a polynucleotide comprising the barcode, a first forward primer binding sequence, and a first reverse primer binding sequence;
• and (iii) a solid support comprising a polynucleotide complementary to at least a portion of an immunoglobulin heavy chain (IgH) mRNA and a polynucleotide complementary to at least a portion of an immunoglobulin light chain (IgL) mRNA:
• (a) reverse transcribing the IgH and IgL mRNAs to form IgH and IgL cDNAs, wherein the IgH cDNA comprises a nucleic acid encoding a heavy chain variable region (VH) and the IgL cDNA comprises a nucleic acid encoding a light chain variable region (VL);
• wherein the IgH and IgL cDNAs comprise a second forward primer binding sequence and a sequence complimentary to the first reverse primer binding sequence;
• and
• (b) amplifying the IgH and IgL cDNAs, wherein the barcode is added to the IgH and IgL cDNAs using a first primer complementary to the first forward primer binding sequence, a second primer complementary to the second forward primer binding sequence, and a third primer complementary to the first reverse primer binding sequence;
• wherein the method further comprises:
• (c) sequencing the amplified barcoded IgH and IgL cDNAs, wherein sequencing the amplified barcoded IgH cDNAs occurs simultaneously with sequencing the amplified barcoded IgL cDNAs.

A method comprising, in at least one vessels of a plurality of vessels, the at least one vessel comprising an immune cell and a first and second solid support, wherein:

• (i) the first solid support comprises a polynucleotide comprising a barcode, a first forward primer binding sequence, and a first reverse primer binding sequence;
• and the second solid support comprises a polynucleotide complementary to at least a portion of an immunoglobulin heavy chain (IgH) mRNA and a polynucleotide complementary to at least a portion of an immunoglobulin light chain (IgL) mRNA;
• (ii) the first solid support comprises (1) a polynucleotide comprising a barcode, a first forward primer binding sequence, and a first reverse primer binding sequence, and (2) a polynucleotide complementary to at least a portion of an IgH mRNA;
• and the second solid support comprises a polynucleotide complementary to at least a portion of an IgL mRNA;
• (iii) the first solid support comprises a polynucleotide complementary to at least a portion of an IgH mRNA;
• and the second solid support comprises a (1) a polynucleotide comprising a barcode, a first forward primer binding sequence, and a first reverse primer binding sequence, and (2) a polynucleotide complementary to at least a portion of an IgL mRNA;
• or
• (iv) the first solid support comprises a polynucleotide complementary to at least a portion of an IgH mRNA;
• the second solid support comprises a polynucleotide complementary to at least a portion of an IgL mRNA;
• and the at least one vessel of the plurality of vessels further comprises a polynucleotide comprising a barcode, a first forward primer binding sequence, and a first reverse primer binding sequence:
• (a) reverse transcribing the IgH and IgL mRNAs to form IgH and IgL cDNAs, wherein the IgH cDNA comprises a nucleic acid encoding a heavy chain variable region (VH) and the IgL cDNA comprises a nucleic acid encoding a light chain variable region (VL);
• wherein the IgH and IgL cDNAs comprise a second forward primer binding sequence and a sequence complimentary to the first reverse primer binding sequence;
• and
• (b) amplifying the IgH and IgL cDNAs, wherein the barcode is added to the IgH and IgL cDNAs using a first primer complementary to the first forward primer binding sequence, a second primer complementary to the second forward primer binding sequence, and a third primer complementary to the first reverse primer binding sequence;
• wherein the method further comprises:
• (c) sequencing the amplified barcoded IgH and IgL cDNAs, wherein sequencing the amplified barcoded IgH cDNAs occurs simultaneously with sequencing the amplified barcoded IgL cDNAs.

A method comprising, in at least one vessel of a plurality of vessels, the at least one vessel comprising (i) an immune cell;

• (ii) a first solid support comprising a polynucleotide complementary to at least a portion of an immunoglobulin heavy chain (IgH) mRNA;
• (iii) a second solid support comprising a polynucleotide complementary to at least a portion of an immunoglobulin light chain (IgL) mRNA;
• and (iv) a third solid support comprising a polynucleotide comprising a barcode, a first forward primer binding sequence, and a first reverse primer binding sequence:
• (a) reverse transcribing the IgH and IgL mRNAs to form IgH and IgL cDNAs, wherein the IgH cDNA comprises a nucleic acid encoding a heavy chain variable region (VH) and the IgL cDNA comprises a nucleic acid encoding a light chain variable region (VL);
• wherein the IgH and IgL cDNAs comprise a second forward primer binding sequence and a sequence complimentary to the first reverse primer binding sequence;
• and
• (b) amplifying the IgH and IgL cDNAs, wherein the barcode is added to the IgH and IgL cDNAs using a first primer complementary to the first forward primer binding sequence, a second primer complementary to the second forward primer binding sequence, and a third primer complementary to the first reverse primer binding sequence;
• wherein the method further comprises:
• (c) sequencing the amplified barcoded IgH and IgL cDNAs, wherein sequencing the amplified barcoded IgH cDNAs occurs simultaneously with sequencing the amplified barcoded IgL cDNAs.