MIBS(Multiwell Incidence-based sequencing) of mLN/PP BCRs
mLN or PP samples were stained as above and sorted at 100 cells per well into 16-32 individual wells in a 96 well plate containing 10 l TCL Buffer (Qiagen) supplemented with 1% b-mercaptoethanol. RNA from single cells or 100-cell pools was reverse-transcribed using oligo-dT as in (Tas, J. M. et al. 2016; Tiller, T. et al. 2009). PCR primers were used as described previously (Mesin, L et al. 2020) with the addition of IgA-specific amplification primers C outer (ATCAGGCAGCCGATTATCAC) and C inner (GAGCTCGTGGGAGTGTCAGTG). Pooled PCR products were then purified using SPRI beads (0.7x volume ratio), gel purified and sequenced with a 500-cycle Reagent Nano kit v2 for single cell libraries and with a 600-cycle Reagent kit v3 for 100-cell pool libraries on the Illumina Miseq platform.