GSM5075999: P6-H2_BCR-seq; Mus musculus; RNA-Seq
All single cell-derived antibodies were expressed separately on the surface of cultured cells. Two fluorescently labeled antigens (H1 and H2) were reacted with the cells, and cells reacting with both H1 and H2, H1 alone, or H2 alone were isolated by FACS. Only the heavy chain of the antibody was amplified by PCR and the sequence was read by Miseq. The antibody sequence in each fraction was determined using the antibody sequence read by single cell as a reference. Cells in each fraction (P5-cros, P6-H2, P7-PR8, P8-inter) were used as template for PCR using primers for heavy chains. After that, adapter primers for NGS were added and the run was performed with Miseq. The CDR3 sequence of the heavy chain, contained in each fraction, was determined. Since we had previously determined the antibody sequence from a single cell, we used that sequence as a reference sequence to identify the antibody sequence contained in each fraction.