title: GSM8717998: sorted CD3- CD56+ cells from tonsillar explant, uninfected; Homo sapiens; RNA-Seq
library_name: GSM8717998
library_construction_protocol: To prepare tonsillar cell suspensions, 12 tissue blocks were placed into 1.5 mL Eppendorf tubes containing 400 µL of RPMI-1640 medium supplemented with 5% FBS (R5 medium). The tissues were mechanically digested using pestles (Labolan) attached to a handheld homogenizer (Labolan). The resulting cell suspensions were filtered sequentially through 70 µm cell strainers (LabClinics) and 30 µm CellTrics® filters (Sysmex) to remove debris. Filtered cells were washed twice with 1X PBS, and residual red blood cells were lysed using ACK Lysis Buffer (Fisher Scientific). Cell count and viability were evaluated with the LUNA Automated Cell Counter (Logos Biosystems). CD56+ cells were enriched from both HIV-infected and uninfected tissue suspensions using the MagniSort™ Human CD56 Positive Selection Kit. After enrichment, the cells were washed and resuspended in 1X PBS for viability assessment with LIVE/DEAD Viability dye, incubated for 20 minutes. Subsequently, the cells were washed, resuspended in a staining buffer (1X PBS with 3% FBS), and labeled with surface antibodies targeting lineage markers. Finally, the stained cells were washed again in staining buffer and resuspended in PBS containing 0.04% BSA. The CD3-CD56+ cell population was sorted from the tonsillar CD56+ cell suspensions of HIV-infected and uninfected tissue blocks using a FACS instrument (Cytek Aurora CS), achieving a final purity of >95% The cellular concentration and viability were verified staining the sample with Trypan Blue (Fisher SC) and evaluated by microscopy. Individual cells were partitioned into Gel Bead‑In‑Emulsions with a Target Cell Recovery of 3478 and 5594 total cells, by using the Chromium X System (10X Genomics). Single cell suspensions were converted to barcoded scRNA-seq libraries using the Chromium GEM-X Single Cell 3' Reagent Kits v4 (10X Genomics) following manufacturer's instructions. After cell lysis and barcoded reverse transcription of RNA, cDNA was amplified during 12 cycles and cDNA QC and quantification were performed on an Agilent TapeStation D5000 DNA ScreenTape (Agilent Technologies). cDNA libraries were indexed by PCR during 15 cycles using PN- 3000431 Chromium Dual Index Kit TT Set A. Size distribution and concentration of 3' cDNA libraries were verified on an Agilent TapeStation D5000 DNA ScreenTape (Agilent Technologies). Finally, sequencing cDNA libraries was carried out on a Nova Seq 6000 sequencer (Illumina) to obtain approximately 40,000 paired-end 150 bp reads per cell.