alias: E-MTAB-6292:Human_Th2_48h_2_p
title: Illumina HiSeq 2500 paired end sequencing; T helper cell type 2 ATAC-seq time-course
design: T helper cell type 2 ATAC-seq time-course
library_construction_protocol: 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.
identifiers.namespace: Sanger Institute / Karolinska Institutet
identifiers.id: E-MTAB-6292:Human_Th2_48h_2_p