The PBMCs were stained with CD45-PE for 30 min at room temperature. Cells were washed twice in FACS buffer (PBS 2% FBS), incubated with Fc block solution for 15 min at room temperature, then each sample labelled with a separate oligonucleotide-conjugated Sample Tag (BD Flex Single-Cell Multiplexing Kits A-D) for 60 min at 4°C. Cells were washed twice in BD Pharmingen Stain buffer. Twelve samples (3500 cells/sample; n=3 from each group - non-SD HW, non-SD OW/OB, SD HW, SD OW/OB) were pooled and loaded onto one BD Rhapsody Cartridge following manufacturer's instructions. The remaining 12 samples were pooled and loaded onto a second BD Rhapsody Cartridge. BD Rhapsody System mRNA Targeted, BD AbSeq, and Sample Tag - 23-24124(02).