title: Smart-seq2 data from memory B cells from an individual living with HIV with  broadly neutralizing plasma and an individual living with HIV who is aviremic without treatment
design: Smart-seq2 data from memory B cells from an individual living with HIV with  broadly neutralizing plasma and an individual living with HIV who is aviremic without treatment
library_construction_protocol: Single IgG+ antigen-specific (HIV-1 Envelope protein reactive) B cells were isolated using a BD FACS-Melody, with the purity threshold set to yield. Cells were sorted one per well into a 96-well plate containing lysis buffer (0.2% Triton X-100 and RNase inhibitor), oligo-DT primers and dNTPs. Nucleic acid extraction cannot be accurately carried out from single cells sorted into individual wells of a 96 well plate. Instead, the Smart-Seq2 protocol was followed to carry out full-length scRNA-seq on single memory B cells, with modification of the pre-amplification step that was optimized to reduce primer-dimer by excluding the IS PCR primers from the PCR mix. Briefly, total mRNA present after collection in lysis buffer in each well was reverse transcribed and then pre-amplified using 18 PCR cycles to generate cDNA that was purified using Ampure XP beads. The purified cDNA was assessed by Agilent Tapestation to confirm a peak at 1-2 kb and was quantified by Qubit for normalisation to 1.5 ng for optimal tagmentation (adjusted from 1 ng to account for the presence of primer-dimer).  Libraries for sequencing were then generated by performing an enrichment PCR of 12 cycles using an Illumina Nextera XT DNA Library Preparation kit with 96 indices, then assessed by Agilent Tapestation to confirm a peak at 300-800 bp and quantified by Qubit for normalisation to 5 nM.