GSM3938201: ATAC.TCell.Donor.B.CSF2neg.2; Homo sapiens; ATAC-seq
Human peripheral blood mononuclear cells were separated from peripheral blood using Ficoll-Paque Plus (GE Healthcare). CD4+ T lymphocytes were further isolated by positive selection using magnetic microbeads (Miltenyi Biotec). Effector Memory T cell subsets were then sorted based on the expression of the following surface markers: CD4+CD25–CD45RA-CCR7-. TEM cells were then PE-labelled for GM-CSF expression with a GM-CSF Cytokine Secretion Assay (Miltenyi Biotec) and sorted for PE+/-. Tagmented DNA from GM-CSF- TEM lysed with Buffer2. 50000 cells were pelleted by centrifugation and re-suspended in 50 µl of cold lysis buffer 1 (10mM Tris-HCl pH 7.4, 10mM MgCl2, 0.1% Igepal CA-630) or 2 (10mM Tris-HCl pH 7.4, 3mM MgCl2, 10mM NaCl, 0.1% Igepal CA-630). After incubation on ice for three minutes, nuclei were pelleted by centrifugation for 20 min at 500g, 4 ºC.  The supernatant was discarded and nuclei were re-suspended in 50 µl of reaction buffer containing 1 µl of Tn5 transposase (made in house), 10 ul of 5x transposase buffer (50mM Tris-HCl, pH 8.4 and 25mM MgCl2), and 39uL of milliQ H2O. The reaction was incubated at 37ºC for 60 minutes with 600 RPM mixing. Then 10 µl of clean up buffer (900mM NaCl, 300mM EDTA), 4ul of 5% SDS and 2 µl of Proteinase K (20 µg/µl) (New England Biolabs) were added and incubated for 30 min at 40 ºC. Tagmented DNA was isolated using SPRI beads (2x) and amplified by PCR. Fragments smaller than 600 bp were isolated using SPRI cleanup and libraries where sequenced on an Illumina NextSeq500 platform.