title: Illumina NovaSeq 6000 paired end sequencing; Transcriptional profiling of human IL-9R+ T helper cells in presence of recombinant IL-9 against untreated control
design: Transcriptional profiling of human IL-9R+ T helper cells in presence of recombinant IL-9 against untreated control
library_construction_protocol: Isolation of T cells from blood: Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Plaque Plus (GE Healthcare, UK) density gradient centrifugation. Human CD4+ T cells were isolated from PBMC by EasySep positive selection kit (Stemcell Technologies) according to manufacturer's instruction. Positively selected CD4+ T cells were washed with PBS and stained for subsequent Th cell subset sorting. Effector memory Th9 (CXCR3-CCR8+CCR6-CCR4+) and Th2 (CXCR3-CCR8-CCR6-CCR4+) were sorted to over 90% purity according to their expression of chemokine receptors from CD45RA-CD25-CD8-CD3+ cells. Single cell Th9 clones were directly sorted into 96-well plate according to their expression of chemokine receptors. After T cell single cell cloning, cells were screened for IL-9R expression by flow cytometry. Isolation of T cells from skin biopsies: Skin biopsies from positive patch test reactions were cultured in culture medium and CD4+ T cells were isolated and screened for IL-9R expression by flow cytometry.    Culture medium consisted of PRMI-1640 with Hepes (GIBCO) supplemented with 5% of heat-inactivated human serum (Swiss Red Cross, Basel, Switzerland), 2mM L-Glutamine (Biochrom), 50U/ml penicillin and 50μg/ml streptomycin (Bioconcept) and 5 IU/ml IL-2 (Roche). T cells were cultured at a density of 0.25-1x105cells/96-well plate in a total volume of 200μL cell culture medium. IL-9R+ T helper cells were incubated with or without IL-9 (5ng/ml) for 12 hours. Total RNA was isolated with RNAeasy kit (Qiagen). Followed Illumina TruSeq Stranded mRNA protocol.