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| Status |
Public on Jun 01, 2015 |
| Title |
DC_MTB_rep4 (Bisulfite-Seq) |
| Sample type |
SRA |
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| Source name |
Monocyte-derived dendritic cells
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| Organism |
Homo sapiens |
| Characteristics |
condition: MTB-infected
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| Treatment protocol |
We infected dendritic cells (DCs) with a Mycobacterium tuberculosis (MTB) strain expressing green-fluorescent protein (H37Rv). Importantly, previous studies have shown that the presence of GFP in this strain does not alter the growth rate or the virulence of the bacilli under axenic conditions, relative to wild-type MTB. M. tuberculosis H37Rv was grown from a frozen stock to midlog phase in 7H9 medium (BD) supplemented with albumin-dextrose-catalase (ADC; Difco).
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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).
DNA from infected and non-infected DCs (6ug) was spiked with 30ng of unmethylated cl857 Sam7 Lambda DNA (Promega, Madison, WI) and further sonicated to an average length of ~100bp using a Covaris ultrasonicator under the following settings for 16 cycles: Duty cycle: 10%; Intensity: 5; Cycles/burst: 100. The sonicated product was further subjected to repair of 3’ and 5’ ends followed by the addition of a non-templated dA-tail before ligation to cytosine-methylated adapters provided by Illumina (Illumina, San Diego, CA) as per manufacturerʼs instructions for genomic DNA library construction. Adapter-ligated DNA of 100-200 bp was isolated by 2% agarose gel electrophoresis, and sodium bisulfite conversion was performed on the resulting sample using the MethylCode™ Bisulfite Conversion Kit (Invitrogen) as per manufacturer’s instructions. Half of the bisulfite-converted, adapter-ligated DNA molecules were enriched by six cycles of PCR with the following reaction composition: 2.5 U of uracil-insensitive PfuTurboCx Hotstart DNA polymerase (Agilent), 5 μl 10X PfuTurbo reaction buffer, 25 μM dNTPs, 1 μl PE Primer 1.0 (Illumina), 1 μl PE Primer 2.0 (Illumina) (50 μl final volume). The thermocycling parameters were: 95 ̊C 2 min, 98 ̊C 30 sec, then 6 cycles of 98 ̊C 15 sec, 60 ̊C 30 sec and 72 ̊C 4 min, ending with one 72 ̊C 10 min step. The reaction products were purified using the QIAquick PCR spin column (Qiagen). Two separate PCR reactions were performed on subsets of the adapter-ligated, bisulfite-converted DNA, yielding two independent libraries from the same biological sample. The quality of the libraries was checked on a Bioanalyzer followed by quantification of the libraries by qPCR using the KAPA Library Quantification Kit prior to sequencing. Samples were sequenced on an Illumina HiSeq 2000 using 50- and 59-bp single-end reads.
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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.
Methylation levels for each CpG site were estimated by counting the number of reported C (‘methylated’ reads) divided by the total number of reported C and T (‘unmethylated’ reads) at the same position of the reference genome using Bismark’s methylation extractor tool. The same strategy was also applied for non-CpG methylation (CHG context, where H is either an A, T, or C nucleotide).
Genome_build: hg19
Supplementary_files_format_and_content: Tab-delimited text files include methylation 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) |
| GSE64177 |
Bacterial Infection Remodels the DNA Methylation Landscape of Human Dendritic Cells (Bisulfite-Seq) |
| GSE64183 |
Bacterial Infection Remodels the DNA Methylation Landscape of Human Dendritic Cells |
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| Relations |
| BioSample |
SAMN03268993 |
| SRA |
SRX818213 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1565945_DC87_MTB_5mC.txt.gz |
109.3 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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