GSM3020265: ATAC-seq on PBMCs from patient CLL2 on day 120 of ibrutinib treatment, CD4 cells; 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
CD4+ T cells
NA
NA
Attributes by original data submitter
Sample
source_name
PBMCs_CLL patient_day 120 of ibrutinib treatment_CD4 cells
library
ATAC-seq
donor_type
chronic lymphocytic leukemia patient
donor_id
CLL2
cell_type
Peripheral blood mononuclear cells (PBMCs)
cell_subtype
CD4+Tcell
treatment_timepoint_days
120
cell_number
50780
processing_batch
ATACTK070
replicate
1
Sequenced DNA Library
library_strategy
ATAC-seq
library_source
GENOMIC
library_selection
other
library_construction_protocol
Patient PBMCs were thawed and washed twice with PBS containing 0.1% BSA and 5 mM EDTA (PBS + BSA + EDTA). Cells were then incubated with anti-CD16/CD32 (clone 93, Biolegend) to prevent nonspecific binding. Single-cell suspensions were stained with combinations of antibodies against CD3 (clone UCHT1), CD4 (clone OKT4), CD5 (clone UCHT2), CD8 (clone SK1), CD14 (clone M5E2), CD19 (clone HIB19), CD20 (clone 2H7), CD24 (clone ML5), CD25 (clone BC96), CD27 (clone O323), CD38 (clone HB-7), CD45RA (clone HI100), CD45RO (clone 304218), CD56 (clone NCAM16.2), CD127 (clone A019D5), CD197 (CCR7, clone G043H7), CD223 (LAG3, clone 11C3C65), CD279 (PD-1, clone EH12.2H7), CD366 (Tim3, clone F38-2E2) and DAPI viability dye (all from Biolegend) for 30 min at 4 °C followed by two washes with PBS + BSA + EDTA. Cells were either acquired with a LSRFortessa (BD) cell analyzer or sort-purified with a MoFlo Astrois (Beckman Coulter). Data analysis was performed with FlowJo (Tree Star) software. We obtained whole blood from three healthy donors with informed consent, and isolated PBMCs through Ficoll-Paque density gradient centrifugation and stored them in liquid nitrogen. Following sample thawing, up to ten million cells were incubated with anti-CD16/CD32 (clone 93, Biolegend) to prevent nonspecific binding. Single-cell suspensions were stained with antibodies against CD3 (clone UCTH1), CD4 (clone OKT4), CD8 (clone SK1), CD19 (clone HIB19), CD20 (clone 2H7), CD24 (clone ML5), CD27 (clone M-T271), CD38 (clone HB-7), IgD (clone 1A6-2) and fixable Zombie Red viability dye (all from BioLegend) for 30 min at 4 °C followed by two washes with PBS + BSA + EDTA. Sorting was conducted using a Sony SH800 cell sorter, pre-gated on live singlet lymphocytes before sorting cell subpopulations as follows: T cell (CD3+), total B cell (CD19+, CD20+), naive B cell (CD27-, IgD+), natural effector B cell (CD27+, IgD+), and memory B cell (CD27+, IgD-). Chromatin accessibility mapping was performed using the ATAC-seq method as previously described (Corces et al, Nature Genetics, 2016), with minor adaptations. In each experiment, a maximum of 50,000 sorted cells were pelleted by centrifuging for 5 min at 4 °C at 300 x g. After centrifugation, the pellet was carefully resuspended in the transposase reaction mix (12.5 µl 2xTD buffer, 2 µl TDE1 (Illumina) and 10.25 µl nuclease-free water, 0.25 µl 5% Digitonin (Sigma)) for 30 min at 37 °C. Following DNA purification with the MinElute kit eluting in 11 µl, 1 µl of the eluted DNA was used in a quantitative PCR (qPCR) reaction to estimate the optimum number of amplification cycles. Library amplification was followed by SPRI size selection to exclude fragments larger than 1,200 bp. DNA concentration was measured with a Qubit fluorometer (Life Technologies). Library amplification was performed using custom Nextera primers (Buenrostro et al, Nature Methods 2013). The libraries were sequenced by the Biomedical Sequencing Facility at CeMM using the Illumina HiSeq 4000 platform and the 50-bp single-end configuration.