GSM7734375: Experiment #2, the 10-day model, CXCR4-tropic, Donor 4; Homo sapiens; RNA-Seq
For the 14-day model, cells were stained with Aqua live/dead stain (Thermo Fisher, Inc., Waltham, MA, USA), and live cells were sorted using FACS Aria or the Sony MA900 Multi-Application cell sorter to separate live cells with robust (CFSElow), moderate (CFSEmed) and no response (CFSEhigh) to the T cell receptor stimulus. CFSElow, CFSEmed and CFSEhigh cells were hashed with TotalSeq™-B0252, TotalSeq™-B0253 and TotalSeq™-B0254 antibodies (Biolegend, Inc., San Diego, CA, USA), respectively. CFSEhigh population was stained for cell maturation markers CD45RA (TotalSeq™-B0063) and CD62L (TotalSeq™-B0147) and an early activation marker CD69 (TotalSeq™-B0146). For 2 of the 3 biological replicates (cells donors 2 and 3), an aliquot of CFSEmed cells was hashed with TotalSeq™-B0255 antibody and stained with isotype controls, TotalSeq™-B0090 and TotalSeq™-B0092. Cell hashing antibodies were diluted 1:8; other antibodies were used undiluted. Antibody staining was performed per Biolegend's protocols. After antibody staining, the cells were mixed back together, 10,000 from each population: CFSElow, CFSEmed, and CFSEhigh, and 500 cells stained with isotype controls, and 12,000 total cells were loaded into the Chromium Controller, aiming to achieve the targeted recovery of 10,000 cells in the scRNA-Seq experiment. For the 10-day model comparing gene expression to the 14-day model, cells were viably frozen and thawed  4 days later, to facilitate occurance of the scRNA-seq experiment on the same day. Cells were stained using Aqua live/dead stain (Thermo Fisher, Inc.) in the end of culture, and live cells were sorted using flow cytometry. Live cells were incubated with antibodies to differentiate cell maturation subsets (CD45RA and CD62L) and activation state (CD69). Additionally, an aliquot of cells from the second biological replicate (donor 2) was hashed at the end of culture with TotalSeq™-B0255 antibody and stained with isotype controls, TotalSeq™-B0090 and TotalSeq™-B0092. The third replicate experiment (donor 2) was conducted in parallel with a mock-infected control, incubated in the same conditions as the latency model, but without the exposure to the virus. This mock-infected control was hashed at the end of culture with TotalSeq™-B0255 antibody and stained with isotype controls, TotalSeq™-B0090 and TotalSeq™-B0092. Cells hashed with isotype controls in replicate experiments 2 and 3 were pooled with the main experimental cells at the ratio of 1:20 before loading into the Chromium Controller. For the 10-day model established using CXCR4- and CCR5-tropic viruses (donors 4-6), fresh CD4+ T cells were used. Biobanked CD4+ T cells from people with HIV (donors 7-9) were viably thawed, stained with Aqua live/dead staining (Thermo Fisher, Inc.) and sorted for live cells. For one of the donors (donor 7), two sequencing reactions were prepared (technical replicate). In all experiments, 12,000 total cells were loaded into the Chromium Controller, aiming to achieve the targeted recovery of 10,000 cells in each scRNA-seq experiment. Reverse transcription to generate cDNA and library preparation for scRNA-Seq were conducted per the manufacturer's instructions (10X Genomics, Inc., Pleasanton, CA, USA). For library preparation, 3' gene expression and 3' feature barcoding library kits (v3 or v3.1) were used. Briefly, CD4+ T cells were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was reversed transcribed to cDNA, which contains an Illumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the appropriate size fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction, Illumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added. Sequencing files generated from the gene expression libraries were named with an abbreviation GEX; sequencing files generated from the feature barcoding libraries were named with an abbreviation FBC.