Single cell 3 solution v3 reagent kit

Viable CD45⁺ cells from each subject purified by FACS were then resuspended at a concentration of 1,000 cells/μl, following the instructions of single-cell 3′ solution v3 reagent kit (10X Genomics) to capture around 10,000 cells per sample. The protoplast suspension was then loaded into Chromium microfluidic chips and barcoded with a 10X Chromium Controller (10X Genomics). RNA from the barcoded cells was subsequently reverse-transcribed, and sequencing libraries were constructed with reagents from a Chromium Single Cell 3′ v3 Reagent Kit (10X Genomics) according to the manufacturer’s instructions. Sequencing was performed with Illumina (NovaSeq 6000) according to the manufacturer’s instructions (Illumina). Each sample was sequenced for at least 150 Gb.

For the scRNA-seq analysis of human blood lymphocytes and NK cells, lymphocytes were sorted as CD45⁺ cells, and NK cells were sorted as CD3/CD19/CD20/CD14⁻CD7⁺ cells using FACS (SONY SH800S); the purity was above 95%. The cells from 4 elderly individuals were pooled in one tube, and the cells from 4 young individuals were pooled in another tube. We then counted and resuspended the pooled cells at a concentration of 1000 cells/μL, aiming for an estimated 6000 cells per library, following the instructions of the single-cell 3′ solution v3 reagent kit (10X Genomics). Single-cell libraries were constructed strictly according to the manufacturer’s standard protocols. Each sequencing library was generated with a unique sample index. Libraries were sequenced on the Illumina NovaSeq 6000 system.