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| Status |
Public on Nov 27, 2024 |
| Title |
mDC 1007 |
| Sample type |
SRA |
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| Source name |
Leukapheresis products
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| Organism |
Homo sapiens |
| Characteristics |
tissue: Leukapheresis products
cell type: mDC
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Leukapheresis products were obtained under an IRB-approved protocol (Institute for Transfusion Medicine, Pittsburgh, PA). Monocyte-enriched, elutriated fractions recovered from 6 normal human donor leukapheresis products were used for generation of DCregs and monophosphoryl lipid A (MPLA; TLR4 ligand low toxicity derivative of bacterial lipopolysaccharide (LPS))-matured sDC. In instances where the elutriated monocyte fraction purity was <90%, CD14-specific immunobeads (Miltenyi, San Diego, CA) were used according to the manufacturer’s protocol to increase monocyte purity to >90%. After 7 days culture, monocyte-derived DCreg and sDC were harvested. The viability of DCregs and sDCs, determined by trypan blue staining, was routinely > 95%. A portion of each DC group was snap-frozen in liquid nitrogen and stored at -80°C for Whole Genome Bisulfate Sequencing (WGBS) DNA Methylation analysis. Portions of the remaining DC were either antibody-stained for key surface molecules, HLA-DR, CD11c, T cell costimulatory and coinhibitory molecules and CD163 (clone RM3/1, Biolegend) and flow cytometry data analyzed using Flow Jo software.
DNA was extracted from frozen cell pellets and underwent bisulfite treatment. The bisulfite modified DNA-sequencing library was generated using the Accel-NGS Methyl-Seq DNA library kit (Swift Biosciences) per the manufacturer’s instructions.
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
Whole Genome Bisulfite Sequencing
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| Data processing |
Low-quality reads and adapter sequences were filtered out by tool Trimmomatic
Given the large file size, surviving reads were further split into subfiles. Reads from each subfile were then aligned to human reference genome hg19 by bisulfite aligner bismark. Then SAMtools was applied to merge the multiple aligned subfiles back into one library and sort files based on chromosomal locations.
Picard tool (http://broadinstitute.github.io/picard/) was employed on the sorted BAM file to mark duplicates.
Methylation calling was performed by bismark methylation extractor to generate a list of chromosomal locations with corresponding methylation depth and rate.
DMR were called by R/Bioconductor package DSS, where the significant DMRs were defined by p-value (p.threshold) = 0.05, minimum region length (minlen) = 30 bps and minimum CpG sites (minCG) = 3.
Assembly: hg19
Supplementary files format and content: TXT file for methylation calling by bismark
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| Submission date |
Oct 03, 2023 |
| Last update date |
Nov 27, 2024 |
| Contact name |
Shuchang Liu |
| E-mail(s) |
[email protected]
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| Organization name |
University of Pittsburgh
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| Street address |
203 Lothrop Street
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| City |
Pittsburgh |
| State/province |
PA |
| ZIP/Postal code |
15261 |
| Country |
USA |
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| Platform ID |
GPL24676 |
| Series (1) |
| GSE244577 |
Epigenetic signature of human vitamin D3 and IL-10-conditioned regulatory DCs |
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| Relations |
| BioSample |
SAMN37668469 |
| SRA |
SRX21982630 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7820731_5-mDCs_1007.sort.mark.bismark.cov.gz |
389.5 Mb |
(ftp)(http) |
COV |
SRA Run Selector |
| Raw data are available in SRA |
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