GSM5525570: D7 (chronic timepoint) anti-CD3/CD28, Donor 3; Homo sapiens; RNA-Seq
Healthy donor PBMCs were obtained with written informed consent at Sloan Kettering Institute, MSKCC, Rockefeller University or ATCC. De-identified blood samples were processed according to the protocols approved by the Institutional Review Board of MSKCC and Rockefeller University.  PBMCs were isolated from peripheral blood using Ficoll-Prep (GE Healthcare) or cell preservation tubes (CPT; Becton Dickinson) according to the manufacturer's instructions and cryopreserved using 90% fetal bovine serum (Gibco) 10% DMSO (Sigma Aldrich) and stored in liquid N2 prior to thawing for in vitro assays.  PBMCs were cultured for 15 hrs or 7 days in RPMI (GIBCO) supplemented with 10% (v/v) heat inactivated FBS, penicillin/streptomycin (100 U/mL), L-glutamine (2 mM), sodium pyruvate (1 mM), nonessential amino acids (0.1 mM), HEPES buffer (10 mM) and 2-mercaptoethanol (50 M) (from GIBCO or Sigma Aldrich) at 37oC 5% CO2 in 96 well plates in the presence of IL2 (GIBCO 100 IU/mL or Roche 1 ng/mL).  Additional conditions included 1 ng/mL TGF (GIBCO).  2 M 5-A-RU was added directly to 50 M methylglyoxal to form 5-OP-RU on Day 1 for in vitro stimulation assays.  5-amino-6-D-ribitylaminouracil  (5-A-RU) was synthesized as previously described (Li et al., 2018), resuspended in 200 M aliquots, cryopreserved, and thawed as needed.  CD3/CD28 beads (Gibco; #11161D) were incubated with PBMCs at a 1:2 bead:cell ratio for scRNA-seq and flow cytometric validation assays. RNA libraries were prepared for sequencing using standard Illumina protocols