Single-cell RNA-seq integrative investigation revealing comprehensive characteristics of HIV-1-infected cells

To comprehensively reveal the characteristics of the cells infected with human immunodeficiency virus type 1 (HIV-1), GFP-expressing HIV-1 was inoculated into a human hematopoietic stem cell-transplanted humanized mouse model. Infected cells (i.e., GFP-positive cells) and uninfected cells (i.e., GFP-negative cells) were respectively sorted and were used for single-cell RNA-Seq analyses. Additionally, CD4+ T cells that were isolated from the mock-infected mice were also analyzed. The single-cell capture, cell lysis, RNA extraction, and cDNA synthesis was performed with Fluidigm C1 system. Prior to cell lysis on the C1 System, the capture site was checked whether it contains a single alive cell. Approximately 10,000 cells were applied to the C1 system (Fluidigm), and 96 cells were captured in the flow cells and separated into independent chambers (small IFC 5-10 µm, Fluidigm). Before the scRNA-Seq library construction, we manually checked each chamber under the microscope. RNA-Seq libraries were constructed according to manufacturers' instructions as follows: first-strand cDNA was synthesized and further amplified using the SMARTer system (Clontech). Illumina sequencing libraries were constructed using Nextera XT DNA Sample Preparation kit (Illumina). The 36-bp single-ended sequencing was performed with illumina HiSeq2500.