In the Turchaninova protocol only the first 15 bps of _IGHG_x or IGHE are non-templated, which results in only 3 positions to distinguish the G and E isotype and none for reliable subtyping of _IGHG_x. This is an effect of primary PCR (not RT) primer location, which I assume was a trade-off between constant segment information and the requirement for proximal primer placement so that the CDR3 is still covered by the shorter one of the reads (100 bp). Two ideas on this:
- Have you looked at Cole et al.? Their protocol was specifically designed to provide constant region information.
- If your sequencing facility could do asymetric 400+160 bp reads (instead of the original 400+100 bp), you could move the constant primers further 3’. We now have a new (Illumina compatible) version of our human Ig single-cell PCR primer set, which I am happy to share. It would provide you with up to 70 bp of the IGH@ constant region, which should be enough for most applications (except IGHG2 vs IGHG4 identification).