title: Mouse_Th2_4h_1
organism: Mus musculus
description: Protocols: T-cells from blood (human) and mouse (mouse). CD4+CD62L+ naive T cells were purified from spleens of wild-type C57BL/6JAX adult (6 - 8 weeks) mice using the CD4+CD62L+ T Cell Isolation Kit II (Miltenyii #130-093-227). Cell culture plates were coated with anti-CD3e antibody (1 ug/ml, eBioscences #16-0031-81) in 1X DPBS (Gibco) at 4°C overnight. Purified naive T cells were seeded at a concentration of 1 M cells/ml on the coated plates in IMDM (Gibco) with 10% heat-inactivated FBS (Sigma), supplied with 5 ug/ml anti-CD28 (eBioscience #16-0281-86) with (Th2) or without (Th0) 10 ng/ml mouse recombinant IL-4 (R&D Systems #404-ML-050). Cells were cultured in plates for up to 72 hours. Mononuclear cells were isolated from the cord blood of healthy neonates at Turku University Central Hospital using Ficoll-Paque PLUS (GE Healthcare, #17-1440-02). CD4+ T cells were then isolated using the Dynal bead-based positive isolation kit (Invitrogen). CD4+ cells from three individual donors were activated directly in 24w plates with plate-bound anti-CD3 (500ng/well, Immunotech) and soluble anti-CD28 (500 ng/mL, Immunotech) at a density of 2×106 cells/mL of Yssel's medium67 containing 1% human AB serum (PAA). Th2 cell polarization was initiated with IL-4 (10 ng/ml, R&D Systems). Cells activated without IL-4 were also cultured (Th0). according to ATAC-seq Experiments were done according to the published protocol with some modification. Briefly, 50,000 cells were washed with ice cold 1X DPBS twice, and resuspended in a sucrose swelling buffer (0.32 M sucrose, 10 mM Tris.Cl, pH 7.5, 3 mM CaCl2, 2 mM MgCl2, 10% glycerol). The cell suspension was left on ice for 10 minutes. Then, a final concentration of 0.5% NP-40 was added, and the cells suspension was vortexed for 10 seconds and left on ice for 10 minutes. Nuclei was pelleted at 500 g at 4°C for 10 minutes. Nuclei were washed once with 1X TD buffer (from Nextera DNA Library Preparation Kit, Illumina, #FC-121-1030), and resuspended in 50 ul tagmentation mix containing: 25 ul 2X TD buffer (Nextera DNA Library Preparation Kit, Illumina #FC-121-1030) 22.5 ul H2O 2.5 ul TDE1 (Nextera DNA Library Preparation Kit, Illumina #FC-121-1030)The tagmentation reaction was carried out on a thermomixer at 37°C, 800 rpm, for 30 minutes. The reaction was stopped by the addition of 250 ul (5 volumes) Buffer PB (from Qiagen MinElute PCR Purification Kit), The tagmented DNA was purified by Qiagen PCR Purification Kit according to manufacturer's instructions and eluted in 12.5 ul Buffer EB from the kit, which yielded ~10 ul purified DNA.The library amplification was done in a 25 ul reaction include: 10 ul purified DNA (from above) 2.5 ul PCR Primer Cocktail (Nextera DNA Library Preparation Kit, Illumina #FC-121-1030) 2.5 ul N5xx (Nextera index kit , Illumina #FC-121-1012) 2.5 ul N7xx (Nextera index kit , Illumina #FC-121-1012) 7.5 ul NPM PCR master mix (Nextera DNA Library Preparation Kit, Illumina #FC-121-1030)PCR was performed as follows: 72°C 5 minutes 98°C 2 minutes [98°C 10 secs, 63°C 30 secs, 72°C 60 secs] x 12 10°C holdAmplified libraries were purified by double Agencourt AMpureXP beads purifications (Beckman Coulter, #A63882). 0.4X beads:DNA ratio for the first time, flow through was kept (removing large fragments); 1.4X beads:DNA ratio for the second time, beads were kept. Libraries were eluted from the beads by elution in 20 ul Buffer EB (from Qiagen PCR Purification Kit).1 ul library was run on a Agilent Bioanalyzer to check size distribution and quality of the libraries.
attributes.tag: ENA first public
attributes.value: 2017-12-14
attributes.tag: ENA last update
attributes.value: 2017-12-14
attributes.tag: External Id
attributes.value: SAMEA104429511
attributes.tag: INSDC center alias
attributes.value: Sanger Institute / Karolinska Institutet
attributes.tag: INSDC center name
attributes.value: Sanger Institute / Karolinska Institutet
attributes.tag: INSDC first public
attributes.value: 2017-12-14T17:03:09Z
attributes.tag: INSDC last update
attributes.value: 2017-12-14T13:48:57Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-6292:Mouse_Th2_4h_1
attributes.tag: age
attributes.value: 6 to 8
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: house mouse
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: individual
attributes.value: 1
attributes.tag: organism part
attributes.value: spleen
attributes.tag: sample name
attributes.value: E-MTAB-6292:Mouse_Th2_4h_1
attributes.tag: strain
attributes.value: C57BL/6J