title: Illumina NovaSeq 6000 sequencing; Transcriptional profiling of human Th9 T cell clones treated with GW9662 against untreated control
design: Transcriptional profiling of human Th9 T cell clones treated with GW9662 against untreated control
library_construction_protocol: Peripheral Blood Mononuclear Cells (PBMC) 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 cells (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 96well plate according to their expression of chemokine receptors.  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 50 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 Th9 cell clones were incubated with GW9662 for 48 hours and activated with αCD3/2/28 for 12 hours  Total RNA was isolated with RNAeasy kit (Qiagen) Illumina TruSeq Stranded mRNA protocol (Directional, first strand)