Bulk BCR Deep Sequencing Protocol

The deep sequencing of bulk BCR was performed using primers and protocols as described previously (27 (link)). Briefly, total RNA from 2 or 3 million peripheral blood mononuclear cells (PBMCs) was extracted (RNeasy Maxi Kit, Qiagen) from each time point (2015 (112 months p.i), 2016 (117 months p.i) and 2018 (138 months p.i)) and antibody sequences were amplified using methods and primers as previously described. The PCR product sizes were verified on agarose gel (E-Gel EX; Invitrogen) and quantified with fluorometry (Qubit; Life Technologies), pooled at approximately equimolar concentrations and each sample pool was re-quantified before sequencing on an Illumina MiSeq (MiSeq Reagent Kit v3, 600-cycle).