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| Status |
Public on Feb 22, 2019 |
| Title |
ATAC-seq on PBMCs from patient CLL5 on day 8 of ibrutinib treatment, CD8 cells |
| Sample type |
SRA |
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| Source name |
PBMCs_CLL patient_day 8 of ibrutinib treatment_CD8 cells
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| Organism |
Homo sapiens |
| Characteristics |
library: ATAC-seq
donor_type: chronic lymphocytic leukemia patient
donor_id: CLL5
cell_type: Peripheral blood mononuclear cells (PBMCs)
cell_subtype: CD8+Tcell
treatment_timepoint_days: 8
cell_number: 75000
processing_batch: ATACTK060
replicate: 1
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| Treatment protocol |
All patients were diagnosed and treated and diagnosed according to the revised guidelines of the International Workshop Chronic Lymphocytic Leukemia/National Cancer Institute. Patients were recruited through clinical trial NCT01804686, aimed at studying the long-term effect of PCI-32765 (ibrutinib) and reflect the clinical and biological heterogeneity of the disease. The study was approved by the ethics committees of the contributing institutions (Semmelweis University and Medical University of Vienna). Informed consent was obtained from all participants.
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| Extracted molecule |
genomic DNA |
| Extraction 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.
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
ATAC-seq on PBMCs from patient CLL5 on day 8 of ibrutinib treatment, CD8 cells
ATAC-seq_CLL5_8d_CD8
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| Data processing |
Base calls provided by the Illumina Realtime Analysis software were converted into BAM format using Illumina2bam and demultiplexed using BamIndexDecoder (https://github.com/wtsi-npg/illumina2bam)
Sequenced reads were trimmed for adaptor sequences
Reads were mapped to hg19 whole genome using bowtie2 v2.2.4 with the –very-sensitive parameter
Duplicate reads were marked and removed with picard tools version 1.118
Peaks for ATAC-seq samples were called with MACS2 version 2.1.1.20160309 using the “–nomodel” and “–extsize 147” parameters
Genome_build: hg19
Supplementary_files_format_and_content: Processed data files in bigWig format contain chromatin accessibility profiles and narrowPeak files contain peak calls for each sample
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| Submission date |
Feb 22, 2018 |
| Last update date |
Feb 22, 2019 |
| Contact name |
Christoph Bock |
| E-mail(s) |
cbock@cemm.oeaw.ac.at
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| Organization name |
CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences
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| Street address |
Lazarettgasse 14
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| City |
Vienna |
| ZIP/Postal code |
1090 |
| Country |
Austria |
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| Platform ID |
GPL20301 |
| Series (2) |
| GSE111013 |
Chromatin mapping and single-cell immune profiling defines the temporal dynamics of ibrutinib drug response in chronic lymphocytic leukemia [ATAC-seq] |
| GSE111015 |
Chromatin mapping and single-cell immune profiling defines the temporal dynamics of ibrutinib drug response in chronic lymphocytic leukemia |
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| Relations |
| BioSample |
SAMN08581229 |
| SRA |
SRX3734551 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3020315_ATAC-seq_CLL5_8d_CD8.bigWig |
76.0 Kb |
(ftp)(http) |
BIGWIG |
| GSM3020315_ATAC-seq_CLL5_8d_CD8.peaks.narrowPeak.gz |
1.4 Mb |
(ftp)(http) |
NARROWPEAK |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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