GSM5509815: Unstim_CD4_SevereAsthma_D26toD31_Gex; Homo sapiens; RNA-Seq
For single-cell RNA-seq (10x Genomics), a maximum of 60,000 total CD4+ T cells from up to 6 donors for resting and stimulation conditions separately and per disease were sorted then pooled into low retention 1.5mL collection tubes, containing 500µL of a 1:1 solution of PBS:FBS supplemented with RNAse inhibitor (1:100). Following sorting, ice-cold PBS was added to make up to a volume of 1400µL. Cells were then centrifuged for 5 minutes (600x g at 4˚C) and the supernatant was carefully removed leaving 5 to 10µL. 25µL of resuspension buffer (0.22µm filtered ice-cold PBS supplemented with ultra-pure bovine serum albumin; 0.04%, Sigma-Aldrich) was added to the tube and the pellet was gently but thoroughly resuspended. Following careful mixing, 33µL of the cell suspension was transferred to a PCR-tube for processing as per the manufacturer's instructions (10x Genomics). Single-cell RNA-seq using 10x genomics platform was performed using Chromium™ Single Cell 3' v3 Reagent Kits following the manufacturer's protocol Libraries were constructed using Chromium™ Single Cell 3' v3 Reagent Kits following the manufacturer's protocol; 100 x 100 bp paired-end reads, 3'TAG, Hg19 human reference genome.