GSM9798604: Sorted T CD4 memory cells, LC11900, Recovered control; Homo sapiens; RNA-Seq
Whole blood was collected from participants in heparin or EDTA-coated tubes, with processing initiated within four hours of collection. PBMCs were isolated from heparin tubes using Ficoll gradient separation and cryopreserved in cell freezing medium (90% heat-inactivated fetal bovine serum with 10% DMSO) until further use. CD4+ T cells were evaluated using an activation-induced marker (AIM) assay. Briefly, cryopreserved PBMCs were thawed, washed and cultured for 24 hours at 37°C and 5% CO2 with SARS-CoV-2 MPs containing spike and non-spike epitopes (1μg/mL), DMSO (negative control), or staphylococcal enterotoxin B (1μg/mL, positive control). Cells were pre-incubated with anti-human CD40 blocking antibody (0.5μg/mL) for 15 minutes prior to stimulation. Following stimulation, cells were stained with fluorochrome-conjugated antibodies, and CD16 and analyzed on a FACSAria IIu cell sorter (BD Biosciences). CD4+ T cells were classified into naïve (CCR7+CD45RA+), central memory (CCR7+CD45RA−), effector memory (CCR7−CD45RA−), and terminally differentiated effector memory (CCR7−CD45RA+) subsets. AIM+ cells were identified as OX40+41BB+. Three populations (naive, total memory, and AIM+) were sorted for subsequent single-cell RNA sequencing, with viability confirmed before experiments. For sorted cells, indexed V(D)J, 5' Feature Barcode, and GEX libraries were prepared using the Chromium Next GEM Single Cell 5' v2 Dual Index kit with Feature Barcoding (10x Genomics). Pools of cells of the same subtype from five to seven individuals were obtained following the Cell Multiplexing Oligo Labeling protocol. Libraries were sequenced on Illumina NovaSeq platform.
GEO
GSM9798604_r1