title: SiteCELL enables on-site PBMCs purification and cryopreservation for immune single cell profiling of diverse ancestries
design: SiteCELL enables on-site PBMCs purification and cryopreservation for immune single cell profiling of diverse ancestries
library_construction_protocol: Two types of biological samples were collected from each participant: (1) a blood sample, at least one 6 ml blood sample was drawn using a lavender-top Vacutainer tube containing EDTA; (2) a saliva sample collected using Oracollect®•DNA OCR-100 swab (DNA Genotek), designed for non-invasive collection of high-quality DNA from oral cells. The swab and blood tube were both labeled with the participant's identification number. For single cell RNA sequencing, cells from different participants were pooled together based on equal cell ratios. To maximize genetic diversity and minimize processing costs, four individuals were selected for each multiplexed library construction. Cryovials from the selected individuals were thawed, and cells were washed before a viability assessment was performed using a microscope-based cell count. The Chromium Single Cell 3' Library & Gel Bead Kit (10x Genomics) was used to generate single cell libraries. Each participant contributed approximately 5,000 cells per multiplex, targeting a recovery of around 20,000 cells per well on the 10x Genomics Chip M. After generating gel bead-in-emulsion (GEM) droplets, the reverse transcription and cDNA amplification was performed within the droplets, incorporating unique molecular identifiers (UMIs) and cell barcodes.  Library preparation followed the standard 10x Genomics protocol, including fragmentation, end repair, A-tailing, adaptor ligation, and PCR amplification. Quality control of the libraries was performed using Qubit® Fluorometry for quantification and Agilent 2100 Bioanalyzer or TapeStation 4150 capillary electrophoresis for size distribution analysis.