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| Status |
Public on Nov 18, 2016 |
| Title |
LPS_d1_H3K9me3 |
| Sample type |
SRA |
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| Source name |
LPS d1 H3K9me3
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| Organism |
Homo sapiens |
| Characteristics |
background strain: Adult Human Peripheral Blood
cell type: Monocyte
treatment: Mono_day1_LPS (LPS exposed for 24 hours, collected at 24 hour of exposure)
chip antibody: Rabbit polyclonal anti-H3K9me3 (Diagenode, cat. # pAb-193-050)
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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 |
Purified cells were fixed with 1% formaldehyde (Sigma) at a concentration of approximately 10 million cells/ml. Fixed cell preparations were sonicated using a Diagenode Bioruptor UCD-300 for 3x 10 minutes (30 seconds on; 30 seconds off). 67µl of chromatin (1 million cells) was incubated with 229µl dilution buffer, 3µl protease inhibitor cocktail and 0.5-1µg of H3K27ac, H3K4me3, H3K4me1, H3K27me3, H3K9me3 or H3K36me3 antibodies (Diagenode) and incubated overnight at 4ºC with rotation. Protein A/G magnetic beads were washed in dilution buffer with 0.15% SDS and 0.1% BSA, added to the chromatin/antibody mix and rotated for 60 minutes at 4°C. Beads were washed with 400µl buffer for 5 minutes at 4°C with five rounds of washes. After washing chromatin was eluted using elution buffer for 20 minutes. Supernatant was collected, 8µl 5M NaCl, 3µl proteinase K were added and samples were incubated for 4 hours at 65°C.Finally samples were purified using Qiagen; Qiaquick MinElute PCR purification Kit and eluted in 20µl EB.
Illumina library preparation was done using the Kapa Hyper Prep Kit. For end repair and A-tailing double stranded DNA was incubated with end repair and A-tailing buffer and enzyme and incubated first for 30 minutes at 20°C and then for 30 minutes at 65°C.Subsequently adapters were ligated by adding 30µl ligation buffer, 10 Kapa l DNA ligase, 5µl diluted adaptor in a total volume of 110µl and incubated for 15 minutes at 15°C.Post-ligation cleanup was performed using Agencourt AMPure XP reagent and products were eluted in 20µl elution buffer. Libraries were amplified by adding 25µl 2x KAPA HiFi Hotstart ReadyMix and 5µl 10x Library Amplification Primer Mix and PCR, 10 cycles. Samples were purified using the QIAquick MinElute PCR purification kit and 300bp fragments selected using E-gel. Correct size selection was confirmed by BioAnalyzer analysis. Sequencing was performed using Illumina HiSeq 2000 machines and generated 43bp single end reads.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
ChIP-seq reads were aligned to the hg19 genome assembly using bwa. For peak calling the BAM files were first filtered to remove the reads with mapping quality less than 15, followed by fragment size modeling (http://code.google.com/p/phantompeakqual-tools/). The peak calling algorithm MACS2 http://github.com/taoliu/MACS/) was used to detect the binding sites for the three studied histone marks at p-value of 10-10. H3K4me1 peaks were called using the broad setting of MACS2 while H3K27ac and H3K4me3 were called using the default (narrow) setting.
Genome_build: hg19
Supplementary_files_format_and_content: bam files sequence and quality, and BigWig files for tracks. All data have been normalized ("Normalized") to a depth per mark except few which have been marked as "NotNormalized"
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| Submission date |
Aug 05, 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 |
GPL11154 |
| Series (2) |
| GSE85245 |
Epigenome maps of time-resolved monocyte to macrophage differentiation and innate immune memory (ChIP-Seq) |
| GSE85246 |
Epigenome maps of time-resolved monocyte to macrophage differentiation and innate immune memory |
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| Relations |
| BioSample |
SAMN05514000 |
| SRA |
SRX2004241 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2262974_9911.H3K9me3.Normalized.bw |
166.3 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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