title: GSM4367497: CD4_AS_AL_CD154Neg_CD137Neg_Batch2; Homo sapiens; RNA-Seq
library_construction_protocol: For single cell RNA-seq assays (10x Genomics), 1000 to 2000 HDM-reactive T cells per subject were collected by sorting in low retention and sterile ice-cold 1.5 mL collection tubes containing 500 µL of PBS:FBS (1:1 vol:vol) with RNAse inhibitor (1:100). HDM-reactive T cells from 6 subjects in each of the 4 groups (asthma with HDM-allergy, asthma without HDM allergy, HDM-allergy without asthma and healthy without HDM-allergy) were collected in the same tube. Collection tubes with ~9,000 to 12,000 sorted cells/study group were inverted a few times, ice-cold PBS was added to reach a volume of 1400 µl, and centrifugated for 5 minutes at a speed of 600 g at 4 ˚C. Supernatant was cautiously removed leaving 5 to 10 µl of volume. Pellets were then resuspended with 25 µl of 10x Genomics resuspension buffer (0.22 µm filtered ice-cold PBS supplemented with ultra-pure bovine serum albumin (0.04 %, Sigma-Aldrich). 33 µl of cell suspension were transferred to an 8 PCR-tube strip for downstream steps as per manufacturer's instructions (10x Genomics, San Francisco). Single-cell RNA-seq using 10x genomics platform was performed using Chromium™ Single Cell 3' v2 Reagent Kits following the manufacturer's protocol Libraries were constructed using Chromium™ Single Cell 3' v2 Reagent Kits following the manufacturer's protocol 100 x 100 bp paired-end reads, 3'TAG, Hg19 human reference genome.
attributes.tag: GEO Accession
attributes.value: GSM4367497