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| Status |
Public on Jun 01, 2015 |
| Title |
DC_NI (ATAC-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).
To prepare nuclei, we spun 100,000 cells at 500g for 5 min, which was followed by a wash using 50 μL of cold 1× PBS and centrifugation at 500g for 5 min. Cells were lysed using cold lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2 and 0.05% IGEPAL CA-630). Immediately after lysis, nuclei were spun at 500g for 10 min using a refrigerated centrifuge. Immediately following the nuclei prep, the pellet was resuspended in the transposase reaction mix (25 μL 2× TD buffer, 2.5 μL transposase (Illumina) and 22.5 μL nuclease-free water). The transposition reaction was carried out for 30 min at 37 °C. Directly following transposition the sample was purified using a Qiagen MinElute kit. Following purification, we amplified library fragments using 25 uL of 2X NEBnext PCR master mix, 0.3 uL of 100X SYBR Green I, 2.5 uL each of Nextera primer index 1 (i7) and 2 (i5), and 10 uL of the transposed DNA, in a final volume of 50 uL. We used the following PCR conditions: 72 °C for 5 min; 98 °C for 30 s; and thermocycling at 98 °C for 10 s, 63 °C for 30 s and 72 °C for 1 min. To reduce GC and size bias in our PCR, we monitored the PCR reaction using qPCR in order to stop amplification before saturation. To do so, we amplified the full libraries for four cycles, after which we took an aliquot (5 ul) of the PCR reaction and added 10 μl of the previous PCR cocktail. We ran this reaction for 19 cycles to determine the additional number of cycles needed for the remaining 45 uL reaction. Libraries were amplified for a total of 9 cycles. The libraries were purified using a Qiagen MinElute kit in 20 μL. Quality of the libraries was verified on a polyacrylamide gel and Bioanalyzer. Libraries were then quantifed by qPCR using the KAPA Library Quantification Kit.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
ATAC-Seq reads were mapped to the human reference genome (GRCh37/hg19) and reads that had a unique alignment and mapping quality of ≥10 were retained for downstream analyses.
genome build: hg19
Supplementary_files_format_and_content: bigwig files were generated from bedgraph files using bedGraphToBigWig (UCSC); Scores represent the number of reads that mapped within a genomic region.
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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 |
GPL16791 |
| Series (2) |
| GSE64173 |
Bacterial Infection Remodels the DNA Methylation Landscape of Human Dendritic Cells (ATAC-Seq) |
| GSE64183 |
Bacterial Infection Remodels the DNA Methylation Landscape of Human Dendritic Cells |
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| Relations |
| BioSample |
SAMN03268947 |
| SRA |
SRX818172 |
| Named Annotation |
GSM1565735_DC234_NI.bw |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1565735_DC234_NI.bw |
229.4 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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