title: GSM2480416: C57BL/6J_Male_NaiveCD4+Tcell_H3K4me3_REP1; Mus musculus; ChIP-Seq
library_construction_protocol: Naïve CD4+ T lymphocytes were purified from single-cell suspensions by MACS depletion of non-CD4+ T cells as well as regulatory T cells and TCRγ/δ+ T cells, using a cocktail of specific biotin conjugated antibodies against CD8a (Ly-2), CD45R (B220), CD49b (DX5), CD11b (Mac-1), Ter-119, CD25 and TCRγ/δ+ using the CD4+ CD62L+ T Cell Isolation Kit II (Miltenyi) to obtain an unlabeled pre-enriched CD4+ T cell fraction. Collected cells were suspended in PBS containing 2% FBS and Fc receptors were blocked with FcR blocking reagent for 10 minutes at 4°C (Miltenyi). Cells were further fluorescently stained with a combination of CD4-FITC (Miltenyi), CD44-APC clone IM7 (eBioscience), CD62L clone MEL-14 (BD Pharmingen), eFluor® 450 CD25 clone PC61.5 (eBioscience), for 10 minutes at 4°C, washed and then stained with DAPI (Life Technologies) to cell sort the live population of naïve CD4+, CD62Lhi, CD44lo, CD25- T lymphocytes using the MoFlo XDP system (Beckman Coulter). Samples were gated on single cells for doublet discrimination and non-viable cells were excluded using the viability dye 4’, 6 – diamidine – 2 – phenylindole (DAPI). AllPrep DNA/RNA Mini Kit (Qiagen) was used to perform simultaneous isolation of RNA and DNA from 106 sorted naïve CD4+ T cells and resting B cells. Purified naïve CD4+ T and B cells were fixed with 1% freshly prepared formaldehyde (Thermofisher) for 10minutes at room temperature with agitation. Fixation was stopped with glycine to final concentration of 125 mM and cells washed three times in ice-cold PBS. 15x106 cells were suspended in 1 mL of lysis buffer (20 mM Hepes, pH 7.6, 1% SDS) supplemented with protease inhibitors (Complete Protease Inhibitor Cocktail, Roche). The chromatin was sheared to an average size of 200bp using a Bioruptor® Pico (Diagenode) with 10 high-energy cycles (30 s on/30 s off) at 4°C and then centrifuged for 5 minutes at 13.000 rpm at room temperature. ChIP was performed on pre-cleared chromatin diluted ten-fold with IP buffer (1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris pH 8, 167 mM NaCl) supplemented with protease inhibitors (Roche). For each immunoprecipitation, reactions of 33 l of chromatin were incubated with each one of the following antibodies: anti-H3K4me3 (1 µg, A5051-001P, Diagenode), anti-H3K36me3 (1 µg, A1847-001P, Diagenode), anti-H3K27ac (1.5 µg, A1723-0040, Diagenode), anti-H3K27me3 (2 µg A1811-001P, Diagenode) and rabbit-IgG (C15410206, Diagenode), overnight at 4°C on a rotating wheel. Chromatin was precipitated with 20μl of a mix of 1:1 protein A to protein G magnetic beads (Dynabeads, Invitrogen) dissolved in IP buffer (containing 0.15% SDS, 0.1% BSA) added to each chromatin-antibody mix and incubated at 4°C for an hour. The complexes chromatin-antibody-beads were washed once in buffer 1 (1 % Triton X-100, 0.1 % SDS, 2 mM EDTA, 150 mM NaCl and 20 mM Tris pH 8), twice in buffer 2 (1 % Triton X-100, 0.1 % SDS, 2 mM EDTA, 500 mM NaCl and 20 mM Tris pH 8) and twice in TE buffer (1 mM EDTA, 10 mM Tris pH 8). The bound fraction was eluted into elution buffer (1 % SDS, 0.1 M NaHCO3) for 20 minutes at room temperature, followed by crosslink reversal (200mM NaCl final concentration) and proteinase K protein digestion for at least 4 hours. DNA was extracted with MinElute PCR Purification Kit (QIAGEN). ChIP-seq libraries for H3K36me3 and H3K4me3 and their respective inputs were prepared at the Wellcome Trust Sanger Institute from ChIP enriched DNA using the NEBnext library prep kit (E6000B-SS) and sequenced using 75bp pair-end reads sequencing on an Illumina HiSeq 2500 platform. ChIP-seq libraries for H3K27me3 and H3K27ac and their respective inputs were prepared at the Radboud University Nijmegen from ChIP enriched DNA using the NEXTflex™ Illumina ChIP-Seq Library Prep Kit from Bioo Scientific and sequenced using 42bp single-end reads sequencing on an Illumina HiSeq 2000 platform.
identifiers.namespace: GEO
identifiers.id: GSM2480416