in vitro culture for 72 hours with anti CD3/28 and Th9-promoting cytokines/antibodies
chip antibody
N/A
Sequenced DNA Library
library_strategy
ATAC-seq
library_source
GENOMIC
library_selection
other
library_construction_protocol
cell isolation protocol: Cells from spleen and lymph nodes were obtained by mechanical disruption. Cells from lung were isolated after incubating lung fragments with 100U collagenase for 1 hour and 10 min, followed by filtering with 100 μm cell strainer. Naïve CD4+/CD25-/CD44lo/CD62Lhi mouse T cells were isolated by cell sorting from spleen and lymph nodes to >95% purity using a FACSAria III Cell Sorter, after magnetic enrichment (StemCell Technologies or Miltenyi Biotec). In vivo Th subsets were isolated from lungs of papain-sensitized mice and further sorted as based on their surface markers. in vitro culture protocol: All in vitro cultured cells were stimulated in RPMI medium with 10% (vol/vol) FCS (Invitrogen), 2 mM glutamine (Invitrogen), 100 IU/ml penicillin (Invitrogen), 0.1 mg/ml streptomycin (Invitrogen), and 20 mM HEPES buffer, pH 7.2–7.5 (Invitrogen), and 2 mM β-mercaptoethanol (Sigma-Aldrich). For in vitro Th cultures, naive T cells were plated at a density of 0.5x106/mL and activated with 10 micrograms/mL plate bound anti-CD3/ anti-CD28 for 72h in the presence of polarizing cytokines and antibodies, with or without 1 micromolar retinoic acid, to promote the differentiation of Th0 (none), Th1 (10ng/mL IL-12, 10 micrograms/mL anti-IL-4), Th2 (20 ng/mL IL-4, 10 micrograms/mL anti-IFN-gamma, Th9 (20ng/mL IL-4, 10 micrograms/mL anti-IFN-gamma, 2.5 ng/mL TGF-beta, 100 units/mL hIL-2), Th17 (10ng/mL IL-6, 10 micrograms/mL anti- IFN-gamma, 10 mg/mL anti-IL-4, 2.5ng/mL TGF-beta), iTreg (10ng/mL TGF-beta, 100 units/mL hIL-2). Papain sensitization protocol: Mice of at least 8 weeks of age were anaesthetized with isoflurane and exposed intranasally to 25 micrograms papain (Calbiochem) in 30 microliters PBS on day 0, 3, 6 and 14. 12-16 hours after the last challenge, cells were isolated from lung Approximately 50,000 cells were isolated, washed with cold PBS, and lysed for 10 minutes in cold lysis buffer (10 mM Tris-HCl [pH 7.4], 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630). After pelleting the nuclei by centrifugation (500×g for 10 min), pellets were resuspended in a 40-ul transposition reaction with 2 ul Tn5 transposase (FC-121-1030; Illumina) to tag and fragmentalize accessible chromatin. The reaction was incubated at 37°C, 400 rpm for 30 min; DNA was then purified using a MinElute kit (QIAGEN) and amplified with 8-12 cycles of PCR based on the amplification curve. After purification using a QIAquick PCR cleanup kit (QIAGEN) and Ampure XP beads (Beckman Coulter) , samples were sequenced for 75 cycles (paired-end reads) on a Illumina HiSeq 2500.