Illumina NovaSeq 6000 paired end sequencing; Single cell RNA sequencing of NK cells from MHC class I knockout (Dd-/-), and H-2Dd transgenic (Dd+/- or Dd+/+) mice
Enriched NK cells from spleens of H-2Dd+/+ and H-2Dd-/- mice were stained with antibodies against NK1.1, CD3, CD19, Ter-119, LY49CI, LY49G2, LY49A, NKG2A and Aqua Live Dead. Cells from each strain were then labeled with different oligo-labeled anti-CD45 antibody for 20 min at 4°C. The experiments were repeated three times with each time having one mouse from each strain. Cells were then sorted using BD FACSAria Fusion (BD Biosciences) to achieve the purity of more than 99 %. Subsequently, cells were pooled and subjected to 10x Genomics' single-cell RNA sequencing, run by Eukaryotic Single Cell Genomics Facility, Science for Life Laboratory, Stockholm. The libraries were prepared with the 10X Genomics Single Cell 3' v3 protocol. The libraries were prepared with the 10X Genomics Single Cell 3' v3 protocol.