GSM5531036: naïve CD4+ T cells sample 3 protected_V(D)J sequence; Homo sapiens; OTHER
Frozen vials of PBMCs were placed a 37°C water bath and gently agitated until ~90% thawed. The vials were transferred to a tissue culture hood and 1mL of sterile warmed media (RPMI/20%FBS) was added to the PBMCs for 5 minutes before transferring drop-wise to a 10ml volume of warmed media (RPMI/20%FBS) containing 200U/ml of DNase I (Roche, Switzerland). After 10 minutes at room temperature, the PBMCs were centrifuged at 300g, at room temperature for 5 minutes. The pellets were washed twice with room temperature media (RPMI/20%FBS) before staining with Live/dead Fixable Aqua (Thermofisher Scientific) for 30 minutes at room temperature. Cells were then washed and incubated with antibodies against CD3, CD4, CD19, IgD, IgM, CD45RO, CD27 and CXCR3 on ice for 30 minutes in MACS buffer (PBS, 2% BSA, 2mM EDTA). Cells were washed, resuspended in a solution of 50% MACS buffer and 50% FBS, and run on the BD Aria III with the 100μm nozzle. Up to 19,000 live naïve CD4+ T cells (CD19- CD3+ CD4+ CD45RO- CD27+) or memory CD4+ T cells (CD19- CD3+ CD4+ CD45RO+) were sorted into solution of 50% MACS buffer and 50% FBS. Sorted cells were applied to the 10x genomics single-cell RNA-seq platform for cell barcoding, cDNA synthesis and 5' gene expression library and V(D)J library preparation following the manufacturer's protocols. 5' gene expression and V(D)J libraries were sequenced on the Hi-seq Illumina platform (2x150bp paired-end, Genewiz). Chromium Next GEM Single Cell V(D)J v1 Single cell RNAseq + Single Cell Immune Profiling