GSM8143812: 4 control subjects and 8 patients with chronic spontaneous urticaria; Homo sapiens; RNA-Seq
Our study sample comprised patients with a clinical diagnosis of CSU under hospital care and healthy controls with no diagnosis of atopic disease or self-reported atopic dermatitis, allergic rhinitis, asthma, food allergy, CSU or spontaneous angioedema. Ethical approval was granted by the Joint Research Ethics Committee in Tallaght University Hospital and St. James's Hospital, Dublin. Using flow cytometry, we assessed the number of Lineage-CD34+CD117+ cells per ml of peripheral blood enumerating cells which were FcεRI negative, FcεRI positive and FcεRIhi. A subset of individuals (n=12) underwent scRNA-seq. Patients completed the Urticaria Control Test (UCT), a measure of disease control assessing hives and swellings over the past 4 weeks (20). For patients who were omalizumab naïve at the time of sampling and went on to receive omalizumab, we sought to compare individuals who responded to omalizumab within 90 days of treatment versus those who did not. We defined 'responders' as individuals with a UCT score ≥ 9, 90 days after their first dose of omalizumab and 'non-responders' as individuals with a UCT score ≤ 8, 90 days after their first dose. 90 days corresponds to 3 doses of 300mg omalizumab received every 30 days. scRNA-seq was performed using the BD Rhapsody Single Cell Analysis System (BD Biosciences). Briefly, sorted cells populations (2 cryopreserved, 10 fresh) were incubated with Human FcX (BioLegend), before labelling cells with antibodies from the Human Single Cell Sample Multiplexing Kit (BD Biosciences). Single cell capture was performed using the BD Rhapsody Express instrument, BD Rhapsody cartridges and the BD Cartridge Reagent Kit. After cell lysis, an on-bead cDNA reaction was performed using the BD Rhapsody cDNA kit. cDNA libraries were prepared using the BD Rhapsody Whole Transcriptome Analysis library preparation kit. Sequencing was performed using an S1 100 cycle Kit on a Novaseq 6000 (with a final sequencing concentration of 300 pM with 20% PhiX (Illumina). Paired-end sequencing was performed with the following cycle allotment: RI 64, R2 58, i7 8, resulting in 1.2 billion quality reads aligning to cells. Bioinformatics was performed using the BD Rhapsody WTA Analysis pipeline (Revision 11). Data was aligned to human genomic sequence and annotation (GRCh38, Release 29). Of a total of 14,964 cells were identified, 244 had an undetermined sample tag call and 2,606 cells were identified as doublets. cDNA libraries were prepared using the BD Rhapsody Whole Transcriptome Analysis library preparation kit.