GSM8447821: Tonsil, Replicate 1, scRNAseq; Homo sapiens; RNA-Seq
Fresh, magnetically enriched cells were washed in MACS buffer and counted to analyze cell viability. Next, cells were resuspended in a cell staining buffer and stained with antibodies for the identification of pDC. All samples were stained with the same antibodies. Specifically, samples were stained with anti-CD3 (PerCP-Cy5.5, Biolegend), anti-CD19 (PerCP-Cy5.5, Biolegend), and anti-CD14 (PerCP-Cy5.5, Biolegend) to exclude T cells, B cells and monocytes, respectively, anti-CD1c (BV510, Biolegend) to exclude mature DC2, and anti-ILT3 (PE-Cy7, Biolegend), anti-CD123 (PE, Biolegend) and anti-BDCA2 (FITC, Millteny) to select pDC. We performed purity checks and achieved a very high purity (>98%). The cells were sorted using flow cytometry on BD FACS Aria II For library preparation, the 10X Genomics Next GEM Single Cell 3' Reagents Kit was used. This process utilized the Chromium Next GEM Single Cell 3′ Kit v3.1 (16 rxns), Chromium Next GEM Chip G Single Cell Kit (48 rxns), and Dual Index Kit TT Set A (96 rxns). In brief, up to 23,000 cells were partitioned into nanoliter Gel-bead-in-Emulsions (GEMs) droplets, each containing retro-transcriptase reaction mix. Single-cell cDNA received a unique 12-nucleotide barcode and unique molecular identifier (UMI). Subsequently, GEM cDNA underwent 11 cycles of amplification before purification using SPRIselect beads. The generation of gene-expression (GEX) and hashtag-oligo (HTO) libraries followed the recommendations provided in the 10x Genomics Chromium Single Cell 3′ Reagent Kits User Guide (v3.1 Chemistry Dual Index), with modifications to PCR cycles based on the calculated cDNA concentration. The concentration of each library was determined using qPCR with the KAPA library Quantification Kit following the manufacturer's protocol (KAPA Biosystems/Roche). The sequencing of normalized cDNA libraries was performed on a NovaSeq6000 S4 Flow Cell using the XP workflow and a 28×10×10×150 sequencing recipe as per the manufacturer's protocol (Illumina). A median sequencing depth of 50,000 and 5,000 reads per cell was targeted for the GEX and HTO library, respectively.