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| Status |
Public on Jun 26, 2016 |
| Title |
ATAC-seq for CLL patient 680, sample 1-5-33943 |
| Sample type |
SRA |
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| Source name |
Primary peripheral blood mononucleated cells (PBMC) from chronic lymphocytic leukemia patient with high leukemic cell count
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| Organism |
Homo sapiens |
| Characteristics |
patient_id: 680
sample_id: 1-5-33943
sample_collection_timepoint: 1
patient_gender: M
ighv_homology: 92
ighv_mutation_status: mutated
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
All patients were diagnosed and treated at the Royal Bournemouth Hospital (UK) according to the revised guidelines of the International Workshop Chronic Lymphocytic Leukemia/National Cancer Institute (IWCLL/NCI). Patients were selected to reflect the clinical and biological heterogeneity of the disease. Sequential samples were included for a total of 24 patients. All samples contained more than 80% leukemic cells.
For ATAC-seq, accessible chromatin mapping was performed using the ATAC-seq method as previously described (Buenrostro 2013, doi:10.1038/nmeth.2688), with minor adaptations. In each experiment, 105 cells were washed once in 50 µl PBS, resuspended in 50 µl ATAC-seq lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, and 0.1% IGEPAL CA-630) and centrifuged for 10 min at 4°C. Upon centrifugation, the pellet was washed briefly in 50 µl MgCl2 buffer (10 mM Tris, pH 8.0, and 5 mM MgCl2) before incubating in the transposase reaction mix (12.5 µl 2x TD buffer, 2 µl transposase (Illumina) and 10.5 µl nuclease-free water) for 30 min at 37°C. After DNA purification with the MinElute kit, 1 µl of the eluted DNA was used in a 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 basepairs. DNA concentration was measured with a Qubit fluorometer (Life Technologies). Library amplification was performed using custom Nextera primers (Buenrostro 2013, doi:10.1038/nmeth.2688). The libraries were sequenced by the Biomedical Sequencing Facility at CeMM using the Illumina HiSeq3000/4000 platform and the 25 basepair paired-end configuration; For RNA-seq, total RNA was isolated using the AllPrep DNA/RNA Mini Kit (Qiagen). RNA amount was measured using Qubit 2.0 Fluorometric Quantitation (Life Technologies), and the RNA integrity number (RIN) was determined using Experion Automated Electrophoresis System (Bio-Rad). RNA-seq libraries were prepared using a Sciclone NGS Workstation (PerkinElmer) and a Zepyhr NGS Workstation (PerkinElmer) with the TruSeq Stranded mRNA LT sample preparation kit (Illumina). Library amount and quality were determined using Qubit 2.0 Fluorometric Quantitation (Life Technologies) and Experion Automated Electrophoresis System (Bio-Rad). The libraries were sequenced by the Biomedical Sequencing Facility at CeMM using the Illumina HiSeq 3000/4000 platform and the 50 basepair single-read configuration; For ChIPmentation, it was carried out as previously described (Schmidl 2015, doi:10.1038/nmeth.3542), with minor adaptions. Briefly, cells were washed once with PBS and fixed with 1% paraformaldehyde in up to 1 ml PBS for 10 minutes at room temperature. Glycine was added to stop the reaction. Cells were collected at 500 x g for 10 minutes at 4°C (subsequent work was performed on ice and used cool buffers and solutions unless otherwise specified) and washed twice with up to 0.5 ml ice-cold PBS supplemented with 1 μM PMSF. The pellet was lysed in sonication buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0, 0.25% SDS, 1x protease inhibitors (Sigma), 1 µM PMSF) and sonicated with a Covaris S220 sonicator for 20-30 minutes in a milliTUBE or microTUBE until the size of most fragments was in the range of 200-700 basepairs. Lysates were centrifuged at full speed for 5 minutes at 4°C, and the supernatant containing the sonicated chromatin was transferred to a new tube. The lysate was then brought to RIPA buffer conditions (final concentration: 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0, 140 mM NaCl, 1% Triton x-100, 0.1% SDS, 0.1% DOC, 1x protease inhibitors (Sigma), 1 µM PMSF) in a volume of 200 µl/immunoprecipitation For each immunoprecipitation, 10 μl magnetic Protein A (Life Technologies) were washed twice and resuspended in PBS supplemented with 0.1% BSA. The antibody was added and bound to the beads by rotating 2 hours at 4°C. Used antibodies were H3K4me1 (0.5 μg/immunoprecipitation, Diagenode pAb-194-050), H3K27ac (1 µg/immunoprecipitation, Diagenode pAB-196-050), H3K27me3 (1 µg/immunoprecipitation, Millipore 07-499). For control libraries an IP with 2.5 µg of a nonspecific IgG rabbit antibody was used. Blocked antibody-conjugated beads were then placed on a magnet, supernatant was removed, and the sonicated lysate was added to the beads followed by incubation for 3-4 hours at 4°C on a rotator. Beads were washed subsequently with RIPA (twice), RIPA-500 (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0, 500 mM NaCl, 1% Triton x-100, 0.1% SDS, 0.1% DOC,) (twice), and RIPA-LiCl (10 mM Tris- HCl, pH 8.0, 1 mM EDTA, pH 8.0, 250 mM LiCl, 1% Triton X-100, 0.5% DOC, 0.5% NP40) (twice). Beads were washed once with cold Tris-Cl pH 8.0 to remove detergent, salts, and EDTA. Beads were washed once more with cold Tris-Cl pH 8.0 but the reaction was not placed on a magnet to discard supernatant immediately. Instead, the whole reaction including beads was transferred to a new tube, and then placed on a magnet to remove supernatant to decrease tagmentation of unspecific chromatin fragments sticking to the tube wall. Beads were then carefully resuspended in 25 μl of the tagmentation reaction mix (10 mM Tris pH 8.0, 5 mM MgCl2, 10% v/v dimethylformamide) containing 1 μl Tagment DNA Enzyme from the Nextera DNA Sample Prep Kit (Illumina) and incubated at 37°C for 1-3 minutes in a thermocycler. The beads were washed with RIPA (twice) and once with cold Tris-Cl pH 8. Beads were washed once more with cold Tris-Cl pH 8.0 but the reaction was not placed on a magnet to discard supernatant immediately. Instead, the whole reaction including beads was again transferred to a new tube, and then placed on a magnet to remove supernatant to remove unspecific tagmented chromatin fragments. Beads were then incubated with 70 μl elution buffer (0.5% SDS, 300 mM NaCl, 5 mM EDTA, 10 mM Tris-HCl pH 8.0) containing 2 μl of Proteinase K (NEB) for 1 hour at 55°C and 8 hours at 65°C to revert formaldehyde crosslinking, and supernatant was transferred to a new tube. Finally, DNA was purified with SPRI AMPure XP beads (sample-to-beads ratio 1:2) or Qiagen MinElute columns. 1 μl of each library was amplified in a 10-μl qPCR reaction containing 0.15 μM primers, 1× SYBR Green and 5 μl Kapa HiFi HotStart ReadyMix (Kapa Biosystems) to estimate the optimum number of enrichment cycles with the following program: 72°C for 5 min; 98°C for 30 s; 24 cycles of 98°C for 10 s, 63°C for 30 s; and 72°C for 30 s; and a final elongation at 72°C for 1 min. Kapa HiFi HotStart ReadyMix was incubated at 98°C for 45 s before preparation of all PCR reactions (qPCR and final enrichment PCR) to activate the hot-start enzyme for successful nick translation in the first PCR step. Final enrichment of the libraries was performed in a 50 μl reaction using 0.75 μM primers and 25 μl Kapa HiFi HotStart ReadyMix. Libraries were amplified for N+1 cycles, where N is equal to the rounded-up Cq value determined in the qPCR reaction. Enriched libraries were purified with size selection using SPRI AMPure XP beads at a beads-to-sample ratio of 1:1, followed by a size selection using AMPure XP beads to recover libraries with a fragment length of 200-400 basepairs. Library preparation was performed using custom Nextera primers as previously described for ATAC-seq (Buenrostro 2013, doi:10.1038/nmeth.2688). The libraries were sequenced by the Biomedical Sequencing Facility at CeMM using the Illumina HiSeq3000/4000 platform and the 50 basepair single-read configuration.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 3000 |
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| Description |
ATAC-seq in primary peripheral blood mononucleated cells (PBMC) from CLL patient 680, sample 1-5-33943
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| Data processing |
Library strategy: ATAC-seq
Illumina Casava1.7 software was used for basecalling.
Sequenced reads were trimmed for adaptor and Nextera 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
Read were extended to the average fragment size and bigWig files containing counts of reads per basepair per million created
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: bigWig files contain counts of reads per basepair per million; narrowPeak files contain peaks called by MACS2
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| Submission date |
May 10, 2016 |
| Last update date |
Jun 26, 2016 |
| Contact name |
Christoph Bock |
| E-mail(s) |
[email protected]
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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 |
GPL21290 |
| Series (1) |
| GSE81274 |
Chromatin accessibility maps of chronic lymphocytic leukemia identify subtype-specific epigenome signatures and transcription regulatory networks |
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| Relations |
| BioSample |
SAMN04967833 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2148395_CLL_ATAC-seq_680_1-5-33943_ATAC30-7.bigWig |
177.8 Mb |
(ftp)(http) |
BIGWIG |
| GSM2148395_CLL_ATAC-seq_680_1-5-33943_ATAC30-7_peaks.narrowPeak.gz |
734.7 Kb |
(ftp)(http) |
NARROWPEAK |
| Raw data not provided for this record |
| Processed data are available on Series record |
| Processed data provided as supplementary file |
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