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| Status |
Public on Jun 01, 2015 |
| Title |
DC_NI (TAB-Seq) |
| Sample type |
SRA |
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| Source name |
Monocyte-derived dendritic cells
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| Organism |
Homo sapiens |
| Characteristics |
condition: Non-infected
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| Treatment protocol |
Dendritic cells (DCs) were infected with a Mycobacterium tuberculosis (MTB) strain expressing green-fluorescent protein (H37Rv) for 18 h at a multiplicity of infection of 1-to-1. Full details can be found in Barreiro et al. (2012).
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| Growth protocol |
Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats by Ficoll-Paque centrifugation. Blood monocytes were then purified from PBMCs by positive selection with magnetic CD14 MicroBeads (Miltenyi Biotech). Pure monocytes were cultured for 5 days in RPMI 1640 (Invitrogen) supplemented with 10% heat-inactivated FCS (Dutscher), L-glutamine (Invitrogen), GM-CSF (20 ng/mL; Immunotools), and IL-4 (20 ng/mL; Immunotools). Cell cultures were fed every 2 days with complete medium supplemented with the cytokines previously mentioned. Before infection, we systematically checked the differentiation/activation status of the monocyte-derived DCs by flow cytometry, using antibodies against CD1a, CD14, CD83, and HLA-DR. Only samples presenting the expected phenotype for non-activated DCs – CD1a+, CD14-, CD83-, and HLA-DRlow – were used in downstream experiments.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA from infected and non-infected DCs was extracted using the PureGene DNA extraction kit (Gentra Systems).
Genomic DNA was spiked with 0.25% of M. SsI methylated lambda DNA and 0.25% of 5hmC spike-in control II (where all cytosines were 5hmC) and then sonicated to 200-500bp with a Covaris ultrasonicator. Next, b-glucosyltransferase (bGT) was used to introduce a glucose onto 5hmC, generating b-glucosyl-5-hydroxymethylcytosine (5gmC) to protect 5hmC from further TET oxidation. After blocking of 5hmC, all 5mC is converted to 5caC by oxidation with an excess of recombinant Tet1 protein. Bisulfite treatment of the resulting DNA then converts all C and 5caC (derived from 5mC) to uracil or 5caU, respectively, whereas the original 5hmC bases remain protected as 5gmC. Thus, following sequencing, only the 5hmC that were protected from bisulfite conversion will be read as cytosine bases. After the treatment, bisulfite conversion and library preparation were performed following a protocol identical to that for MethylC-Seq libraries.
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Adapter sequences and low quality score bases (Phred score < 20) were first removed from reads.
Trimmed reads were mapped in bisulfite mode to human reference genome (GRCh37/hg19) and control sequences and PCR duplicates were removed.
Hydroxymethylation levels for each CpG site were estimated by counting the number of reported C (‘hydroxymethylated’ reads) divided by the total number of reported C and T (‘non-hydroxymethylated’ reads) at the same position of the reference genome.
Genome_build: hg19
Supplementary_files_format_and_content: Tab-delimited text files include hydroxymethylation levels for each covered CpG site for each sample.
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| Submission date |
Dec 15, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Alain Pacis |
| E-mail(s) |
alain.pacis@mcgill.ca
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| Organization name |
Canadian Centre for Computational Genomics (C3G)
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| Street address |
740 Dr Penfield Ave
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| City |
Montreal |
| State/province |
QC |
| ZIP/Postal code |
H3A 0G1 |
| Country |
Canada |
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| Platform ID |
GPL11154 |
| Series (2) |
| GSE64181 |
Bacterial Infection Remodels the DNA Methylation Landscape of Human Dendritic Cells (TAB-Seq) |
| GSE64183 |
Bacterial Infection Remodels the DNA Methylation Landscape of Human Dendritic Cells |
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| Relations |
| BioSample |
SAMN03268959 |
| SRA |
SRX818220 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1565996_DC82_NI_5hmC.txt.gz |
111.1 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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