GSM3938193: ATAC.TCell.Donor.A.CSF2pos.2; Homo sapiens; ATAC-seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
ATAC-Seq
Antigen
ATAC-Seq
Cell type
Cell type Class
Blood
Cell type
PBMC
Tissue
blood
Lineage
mesoderm
Description
peripheral blood mononuclear cells
Attributes by original data submitter
Sample
source_name
primary T lymphocytes derived from human peripheral blood mononuclear cells (PBMC)
cell type
Effector memory T lymphocytes
label
PEpos
antibody
none
Sequenced DNA Library
library_strategy
ATAC-seq
library_source
GENOMIC
library_selection
other
library_construction_protocol
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.