CD3+ CD4+ T cells were sequentially isolated from PBMCs of aGCA or HD using Dynabeads Untouched Human T Cells kit and Dynabeads CD4+ isolation kit (Thermofisher Scientific). Sample purities were assessed afterwards and were all > 95%. Total RNA from CD4+ T cells was then extracted using the NucleoSpin RNA kit (Macherey-Nagel) and quantified by a NanoDrop 1000 spectrophotometer. 5 samples with RNA concentration < 20ng/µL were excluded. For quality control, RNA dilution was performed using Agilent RNA 6000 Nano Kit and 1µL of the sample was run on the Nano chip using an Agilent 2100 electrophoresis bioanalyzer. The quality of total RNA was assessed using the profile of the electropherogram and the RNA integrity number (RIN) was calculated. The whole samples showed RINs between 7.1 and 9.5. For Illumina Beadarrays, cRNA samples were prepared using Illumina TotalPre-96 RNA Amp kit (LifeTechnologies) and hybridized to Illumina Human HT-12 v4 beadarrays.
Label
Cy3
Label protocol
not provided
Hybridization protocol
not provided
Scan protocol
not provided
Data processing
Then, raw IDAT files were processed using illuminaio Bioconductor R package and concatenated into a single text file. Data were further background-corrected using limma Bioconductor R package and inter-chip batch effects were removed using ComBat method from sva Bioconductor R package. In the end, the following samples numbers remained and were analyzed: 15 CD4+ T cells from HD and 19 CD4+ T cells from aGCA.
