GSM8579968: HYST114 HIV; Homo sapiens; OTHER
Tissues from the endometrium and ectocervix were obtained after hysterectomy. Tissues were minced into 1-2 mm fragments in Roswell Park Memorial Institute (RPMI) medium (Gibco) containing enzymes from Tumor Dissociation Kit, human (Miltenyi Biotec) and 0.01% DNAse (Worthington Biochemical) and transfered into sterile gentleMACSTM C Tubes (Miltenyi Biotec). Enzymatic digestion was performed on genlteMACSTM Dissociator (Miltenyi Biotec) using “37C_h_TDK_1” program. Digested tissue was filtered through 100um, 70um and 30um MACSTM SmartStrainers (Miltenyi Biotec) to generate stromal single cells suspensions. Single-cell suspensions were enriched for immune cells by magnetic bead removal of CD235a+ (CD235a (Glycophorin A) MicroBeads, human; Miltenyi Biotec) red blood cells and fibroblasts (Anti-fibroblast MicroBeads, human; Miltenyi Biotec). Purified cells were washed thoroughly with PBS containing 0.5% HS to remove any excess magnetic beads. The cells were incubated with HIV-BaL (MOI = 0.5) in XVIVO-15 or media alone for 30 minutes at 37C after which they were washed to remove excess unbound virus. Subsequently, the cells were incubated with oligo-conjugated antibodies (AbSeq) and barcoded sample tags (BD Hu Single Cell Sample Multiplexing Kit; BD Biosciences), to differentiate between tissue sites, for 20 minutes at room temperature in PBS containing 0.5% HS. Single cells from control and HIV treated samples were subsequently captured on separate cartridges, followed by synthesis of cDNA on BD Rhapsody Express system according to manufacturer's protocol https://www.bdbiosciences.com/content/dam/bdb/marketing-documents/Protocol-Enh-Bead-WTA-%2B-AbSeq-%2B-SMK-RUO.pdf Libraries containing whole transcriptome, protein (AbSeq) and sample multiplexing (SampleTag) were constructed from generated cDNA according to manufacturer's protocol. https://www.bdbiosciences.com/content/dam/bdb/marketing-documents/Protocol-Enh-Bead-WTA-%2B-AbSeq-%2B-SMK-RUO.pdf