• I'm trying to sequence the variable regions of immunoglobulin G based on existing methods (PMID: 24114719, PMID: 30779032) using cultured single cell plasma cells as the mRNA substrate
• At day 3 in culture, cells are are lysed, RNA is purified through a single cell RNA purification kit and undergo RT-PCR and then nested PCR as per the above protocols (performed separately for heavy and kappa/lambda templates)
• The curiosity is that when I do this and send the PCR products for Sanger sequencing using specific primers, I usually obtain either the heavy chain sequence or the the light chain sequence, but never both from the same cell
• The PCR reactions are performed separately so it can't be due to polymerase/primer competition. I understand that light chains are more abundant and therefore should be easier to sequence, but surely that implies that if the heavy chain mRNA were of sufficient quality/quantity to sequence, so should be the light chains in the same protocol?

Any thoughts would be greatly appreciated!