Easysep human cd8 positive selection kit 2

CD8+ T lymphocytes were isolated from PBMCs using the EasySep™ Human CD8 Positive Selection Kit II on a STEMCELL EasyEights magnet following the manufacturer’s instructions. After isolation, the CD8+ T lymphocytes obtained were quantified, and the degree of purity was above 80% (CD3-BV605 clone SK7 and CD8-V450 clone RPA-T8). An RNeasy Plus Mini Kit (Qiagen, Valencia, CA, USA) was used to extract total RNA from the samples following the manufacturer’s recommendations. RNA levels were measured using the NanoDrop ND-1000 spectrophotometer (Thermo Scientific, MA, USA). For cDNA synthesis, an RT2 First Strand kit (Qiagen) was used.
For real-time PCR reactions, an RT2 SYBR Green/ROX qPCR Master Mix (Qiagen) was used, which contains SYBR Green as a fluorophore and ROX as a passive reference. The PCR array kit used was PAHS122Z Antiviral Response (Qiagen) in accordance with the manufacturer’s instructions. Gene expression data from purified CD8+ T lymphocytes were analyzed using the comparative Ct method. For normalization of the data, an average value of the following reference genes was used: ACTβ (beta-actin), GAPDH (glyceraldehyde 3 phosphate dehydrogenase), HPRT1 (hypoxanthine phosphoribosyl transferase 1), and RPLP0 (ribosomal protein, large, P0).

Peripheral blood mononuclear cells (PBMCs) were isolated from human blood by Ficoll density gradient centrifugation. For adult donors in the cross-sectional cohort, CD8⁺ cells were enriched from PBMCs using an EasySep Human CD8 Positive Selection Kit II (STEMCELL Technologies) according to the manufacturer’s instructions. Cells were resuspended in phosphate-buffered saline (PBS) containing 0.04% bovine serum albumin (BSA). Purity was over 95% for all samples. PBMCs from cord blood were cryopreserved and thawed on the day of use in warm media for 1 h, washed once with BSM (HBSS containing 0.2% BSA, 1X HEPES, 1X penicillin-streptomycin-glutamine), and stained with fluorescent antibodies against CD3, CD4, and CD8. CD8⁺ cells were sorted as CD8⁺/CD4^- cells into PBS containing 0.04% BSA. For longitudinal cohort samples, cryopreserved PBMCs were thawed on the day of use in warm media for 1 h, washed once with BSM, and stained with antibody cocktails containing fluorescent antibodies against CD8 and CD4, and DNA barcoded antibodies against CD28 and CD45RA (Supplementary Fig 1c). CD8⁺ cells were sorted as CD8⁺/CD4^- cells into PBS containing 0.04% BSA. Purities of CD8⁺ T cells ranged from 97% to 98%. For scRNA-seq library construction, cells were diluted to ~1 ×10⁶/mL in 100 µL PBS containing 0.04% BSA and processed within hours after they were obtained.