gp120neg B cells;Blood;Month 0: Sample M28S21
For the Ig VH sequencing library preparation, total RNA from B cells was isolated and treated with DNase I, and ~1/9 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 forward (VH1-6) primers from framework region (FR)1 and reverse (IgA, IgG and/or IgM isotype-specific) primers from constant domain 1, followed by a 10 cycles PCR for addition of respective Illumina Nextera sequencing tags. 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 2 x 300 bp paired-end kits.
M28S21