Single-cell BCR-Seq of Homo sapiens: adult
PBMCs were isolated using the Lonza lymphocyte separation medium and cryopreserved until ready for processing. PBMCs were then thawed, incubated with DNaseI, washed, and resuspended in PBS. 2 million PBMCs (~150,000 B cells) were then run through the custom made Flow Focusing Device to encapsulate single cells in oil and water emulsions containing oligo-d(T) magnetic beads and lysis buffer. Within the emulsions, mRNA was captured following cell lysis. Emulsions were then broken using diethyl ether, and the magnetic oligo-d(T) beads were captured, washed, and resuspended in overlap-extension (OE)/RT-PCR mix. Single beads were then re-emulsified and  BCR heavy and light chain mRNA transcripts from single cells were physically linked, reverse-transcribed, and amplified within the emulsions.