Cd56 pe cy7 ncam16.2

Cryopreserved PBMC were thawed in media containing 20% fetal bovine serum. Cell counts and viabilities were assessed using Guava ViaCount reagent and a Guava PCA (Millipore Sigma). Cells were washed and stained with Aqua Live/Dead stain (Molecular Probes), washed, and blocked using normal mouse IgG (Caltag). The cells were surface stained for CD3 FITC (clone UCHT1, Becton Dickinson [BD]), CD8 PerCP-eF710 (SK1, eBiosciences), CD14 V500 (M5E2, BD), CD45RO eF650NC (UCHL1, eBiosciences), CD4 APC (RPAT4, BD), CD45RA APC-H7 (HI100, BD), CD19 PE-Cy5 (SJ25-C1, Invitrogen), and CD56 PE-Cy7 (NCAM16.2, BD) (Figure S2). The cells were then washed and sorted/analyzed on the BD FACSAria II SORP Cell Sorter. Total RNA from sorted cell subsets was extracted using the Single Cell RNA Purification kit (Norgen Biotek Corp). RNA was prepared for NGS RNA sequencing (RNAseq) using the SMART-Seq technology as described previously (Ehrenberg et al., 2019 ; Picelli et al., 2014 (link); Ramsköld et al., 2012 (link); Shangguan et al., 2021 ). Briefly, cDNA was generated per manufacturer’s instructions from 2.5 ng of RNA, final exogenous ERCC RNA spike-in dilutions of 1:190,000, and 11 cycles of library PCR amplification. A total of 300 ng of cDNA was input template for final library processing using the Nextera XT kit (Illumina) per manufacturer’s instructions.