gp120pos B cells;Blood;Month 0: Sample M25S03
total RNA from PBMCs, plasmablasts and bone marrow was isolated and treated with DNase I, and ~1/10 part of this RNA was used for cDNA synthesis in a 20 μL reaction with the qScript cDNA synthesis kit (QuantaBio, MA, USA). A 45 cycles PCR was carried out in a 50 µl reaction with 5 µl of the resulting cDNA as template with a cocktail of illumina linker tagged forward (VH1-7) from framework region (FR)1 and reverse (IgA, IgG and/or IgM isotype-specific) from constant domain 1.  Following the PCR, amplicons were analyzed in 1 % agarose gel and bands corresponding to approximately 600 bp were purified by gel extraction and sequenced on an Illumina MiSeq system (Illumina, Inc., CA, USA) using 300 × 325 bp paired-end kits.
M25S03