GSM9376321: DPNK-1; Homo sapiens; RNA-Seq
NK cells were isolated from thawed CBUs after purification of CBMCs via Ficoll gradient centrifugation, as described above. The isolated NK cells were then subjected to FACS to obtain purified NK cell subsets. First, Fc receptors were blocked using Human TruStain FcX (BioLegend; 1:50) for 15 min at 4 °C. Cells were then stained for 30 min at 4 °C  with the following fluorochrome-conjugated antibodies: anti-human CD45-Krome Orange (Beckman Coulter; 1:40), DAPI (ThermoFisher; 1:1000), anti-human CD56-BV510 (Biolegend; 1:200), anti-human CD16-FITC (Biolegend; 1:100), anti-human CD161-APC (Miltenyi Biotec; 1:100), anti-human CD3-PE (Biolegend; 1:200), anti-human CD33-PE (Biolegend; 1:200), anti-human CD14-PE (Biolegend; 1:200). Live CD56+CD3-CD33-CD14- NK cells were sorted to subsets expressing both CD16 and CD161, denoted as the double positive (DP) NK cell subset; or subsets that lacked both CD16 and CD161 expression, denoted as the double negative (DN) NK cell subset. Sorted NK cell subsets were expanded by co-culture with uAPCs and exogenous IL-2 (200 U/ml) for two weeks. DP/DN-derived NK cells were then stained with combinations of the following nucleotide barcode-labeled antibodies (BioLegend) for epitope labeling: TotalSeq-C1290 anti-human CD3 (HIT3a; 1:200), TotalSeq-C0168 anti-human CD57 (QA17A04; 1:200), TotalSeq-C0166 anti-human CD66b (6/40c; 1:200) , TotalSeq-C0100 anti-human CD20 (2H7; 1:200), TotalSeq-C0083 anti-human CD16 (3G8; 1:200), TotalSeq-C0051 anti-human CD14 (63D3; 1:200), TotalSeq-C0050 anti-human CD19 (HIB19; 1:200), TotalSeq-C0251 anti-human Hashtag 1 (2M2; 1:200), TotalSeq-C0252 anti-human Hashtag 2 (2M2; 1:200), TotalSeq-C0253 anti-human Hashtag 3 (2M2; 1:200), TotalSeq-C0254 anti-human Hashtag 4 (2M2; 1:200), TotalSeq-C0052 anti-human CD33 (P67.6; 1:150), TotalSeq-C0048 anti-human CD45 (2D1; 1:150), TotalSeq-C5100-custom-CD161 (REA631; 1:150), TotalSeq-C0047 anti-human CD56 (5.1H11; 1:150), TotalSeq-C0149 anti-human CD161 (HP-3G10; 1:150). As described in the GEM-X Flex gene expression protocols provided by the manufacturer (10x Genomics), cells were fixed with fixation buffer overnight in 4°C. Fixation was quenched with additive C and quenching buffer. Cells were then washed twice with quenching buffer and counted, 500,000 cells from each sample were loaded. Libraries were prepared using the GEM-X Flex Gene Expression Reagent Kit (10x Genomics, Chromium Human Transcriptome Probe Set v1.1.0) following the manufacturer's instructions. Briefly, each sample was subjected to probe hybridization for their gene expression (GEX) and TotalSeq antibody-derived epitope tags (ADT), respectively. Samples were then pooled for GEM generation and library preparation of both GEX and ADT fractions. Library size and quality were determined using a Fragment Analyzer (Agilent TapeStation Traces) before fragmentation and after final purification. Final library concentrations were measured on a Qubit 2.0 fluorometer (Thermo Fisher). For each sequencing reaction, pooling was performed with the aim to yield ~30,000 reads per cell for the GEX libraires and ~10,000 reads per cell for the ADT libraries. The pooled GEM-X Flex libraries were sequenced on the Illumina NovaSeq X Plus instrument using R1-i7-i5-R2 parameters of 28+10+10+90 cycles.