V1 chemistry

For all 42 individuals tested in MGIA, both at pre and post-vaccination samples were generated for scRNAseq from exactly the same vial of PBMCs. Samples were generated by stimulation of 5 × 10⁵ rested PBMCs for 4 h with 10 ng/mL LPS (serotype 055: B5; Sigma) or left unstimulated at 37 °C, 5% CO₂. LPS was selected for restimulation as heterologous innate immune activator. Upon stimulation, cells were washed in PBS and pooled based on the individual donor, time point of vaccination, and stimulation condition. Pools contained amaximum of 5 samples. As the scRNAseq samples originated from the same vial as the MGIA was performed, the batches processed for sequencing related to the three independent experimental runs for the MGIA. Each MGIA run yielded 10–12 pools of scRNAseq samples. In total 168 samples were processed for single-cell RNAseq at the Leiden Genome Technology Center, Leiden University Medical Center, Leiden, The Netherlands (Supplementary Fig. 1). Briefly, single-cell gene-expression libraries were generated on the 10x Genomics Chromium platform using the Chromium Next GEM Single Cell 3′ Library & Gel Bead Kit v3.1 and Chromium Next GEM Chip G Single Cell Kit (10x Genomics) according to the manufacturer’s protocol. Libraries were sequenced on a NovaSeq 6000 S4 flow cell using v1 chemistry (Illumina) with 28 bp R1 and 90 bp R2 run settings.