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| Status |
Public on Nov 18, 2016 |
| Title |
ATAC_RPMI_d6_8454 |
| Sample type |
SRA |
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| Source name |
Adult Human Peripheral Blood
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| Organism |
Homo sapiens |
| Characteristics |
cell type: Monocyte
treatment: Culture only, collected at 6 days post culture.
tissue: Peripheral blood
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| Growth protocol |
Monocytes were differentiated into resting macrophages by ex vivo culture in RPMI 1640 medium (Sigma Aldrich) with 10% Human Serum. Tolerization was induced by treatment of monocytes with 10-100ng/mL LPS for 24 hours, followed by washout and five days culture in RPMI + 10% human serum, while trained innate immunity was induced by treatment with 5 ng/mL BG for 24 hours, followed by washout and 5 days in culture. Establishment of tolerance or training in the resulting macrophages at day 6 was determined by TNF and IL6 release at 24 hours following LPS stimulation using ELISA. For ChIP-seq, 10x106 monocytes were seeded in 10cm dishes, for RNAseq and ATAC-seq 1.5 x 106 monocytes were seeded in 6 well plates.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Monocytes or macrophages (100,000 cells) were scrapped in a well of a 6-well plate with cold PBS and then spun down at 800 x g for 5 min at 4 ˚C. Cells were washed with 50 µL of cold 1x PBS buffer, incubated in 50 µl of cold lysis buffer (10 mM Tris-HCL (pH 7.4), 10 mM NaCl, 3 mM MgCl2 0,1 % IGEPAL) and spun down at 800 x g for 10 min at 4˚C. The nuclei were immediately resuspended in the transposition reaction mix (22.5 µL TD buffer, 2.5 µL Tn5 Transposase, 25 µL NF H2O) and incubated for 30 min at 37˚C. Following transposition, 100 µL AMPure beads were added to the reaction (sample-to-bead ratio of 1:2), mixed thoroughly by pipetting, and incubated for 15 min at RT. Samples and beads were washed on the magnetic rack with 80% ethanol, dried for 5 min, and resuspended in 15 µl EB buffer. DNA was amplified with 10 - 15 PCR cycles using the mix (15 µL transposed DNA, 0.3 µL 100x SYBR Green I, 25 µL NEBNext High-Fidelity master mix, 2.5 µL Nextera Primer index N7.. (25 µM), 2.5 µL Nextera Primer index S5.. (25 µM), 4.7 µL NF H2O). In order to reduce GC and size bias in PCR, the PCR reaction is monitored using qPCR to stop amplification prior to saturation. Following amplification, samples were incubated purified twice using SPRI beads, first using negative selection with a sample-to-bead ratio of 1-0.65 and then positive selection with a sample-to-bead ratio of 1-1.8. After 80% Ethanol wash and drying, the sample was eluted in 20 µl EB buffer, and quality checked before sequencing. Detailed protocol can be found on the Blueprint website (http://www.blueprint-epigenome.eu/UserFiles/file/Protocols/ATAC_Seq_Protocol.pdf).
Library was constructed using the Nextera kit.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Library strategy: ATAC-seq
Illumina Casava software 1.8.2 used for basecalling of NextSeq 500 data.
Library strategy: ATAC-seq. Paired-end reads were mapped to human reference genome hg19 using bwa mem. Reads with mapQ>=30 were kept for downstream analysis. Peak calling was done for individual samples using macs (version 2.0.10.20130306) with “-g hs --nomodel -p 1e-10”.
Genome_build: hg19
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| Submission date |
Sep 22, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Hendrik G Stunnenberg |
| E-mail(s) |
H.Stunnenberg@ncmls.ru.nl
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| Phone |
+31 (0)24 3610524
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| Organization name |
Radboud University
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| Department |
Molecular Biology
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| Street address |
Geert Grooteplein 28
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| City |
Nijmegen |
| ZIP/Postal code |
6525 GA |
| Country |
Netherlands |
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| Platform ID |
GPL18573 |
| Series (2) |
| GSE85246 |
Epigenome maps of time-resolved monocyte to macrophage differentiation and innate immune memory |
| GSE87218 |
Epigenome maps of time-resolved monocyte to macrophage differentiation and innate immune memory (ATAC-Seq) |
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| Relations |
| BioSample |
SAMN05804998 |
| SRA |
SRX2185367 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2325691_ATAC_RPMI_d6_8454.bw |
432.1 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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