title: GSM8715452: Donor 2 survivor TCRab after CTL co-culture; Homo sapiens; OTHER
library_construction_protocol: Cryopreserved infected CD4+ T-cells were thawed for 1-2 minutes in a 37o C water bath, washed twice RPMI 1640 supplemented with 10% FBS. Infected cells were then rested for 2 days at 2 x 106 cells per mL in media containing 50 IU of human IL-2.  Prior to co-culture with CTL, cells expressing high levels of CD4 were depleted by magnetic selection using the EasySep™ Human CD4 Positive Selection Kit II (Stemcell); this step enriches for CD4- infected cells (since HIV-infected cells downregulate CD4). Cells infected with WT HIVJRCSF were labeled with CFSE (dilution 1:5,000) and cells infected with HIVJRCSF TW10escape were labeled with CTV (dilution 1:10,000). These were then mixed together before adding a TW10-specific CTL that had been labeled with CellTrace™ Blue CTB (dilution 1:10,000). No CTL control conditions were cultured in parallel. Post-killing, cells were harvested for staining with anti-CD4 BV785 (1:100), Live/Dead fixable Aqua (1:200), bNAbs PGT121 conjugated with Zenon Kit PE (1µg per 1 x 106 cells) and bNAbs N6/PGDM1400x10E8 conjugated with Zenon Kit AF647 (1µg per 1 x 106 cells) at 37C for 1h. Live cells productively infected CFSE-labeled WT HIVJRCSF and CTV-labeled HIVJRCSF TW10escape were sorted by gating on the double positive population for PGT121 and N6/PGDM1400x10E8. All experiments were performed using the 10x genomics V(D)J solution which incorporates scRNA-seq with additional profiling of protein surface markers . Antibody oligo counts are listed in the ADT and HTO tables. Stained cells from each sample were pooled and loaded into the 10X Chromium Single Cell Immune Profiling workflow according to the manufacturer's instructions (CG000330, revision G)