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| Status |
Public on Apr 29, 2019 |
| Title |
OxBS_WT_72h |
| Sample type |
SRA |
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| Source name |
Tet2/3-WT_72h_oxBSseq
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
age: 8-12 weeks
genotype/variation: Tet2/3-WT (Tet2-flox, Tet3-flox, Rosa26-LSL-YFP)
timepoint: 72h
tissue: Spleen
molecule subtype: Oxidative bisulfite-treated genomic DNA
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| Treatment protocol |
Cells were either unstimulated or activated with LPS (25ug/mL) and IL-4 (10ng/mL)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was purified using DNaeasy Qiagen blood and tissue kit (Qiagen). Three PCR products containing C, mC, or hmC pertaining to different regions of ʎ phage genome were used as spike-ins at a ratio of 1:200 of the genomic DNA. 1.5 µg of genomic DNA mixed with spike-ins was precipitated with ethanol and 1 µg of the precipitated DNA was oxidized with potassium perruthenate (KRuO4; Sigma) followed by bisulfite treatment (for oxBS). As control, 0.5 µg of DNA was directly used for bisulfite treatment.
The BS and oxBS treated DNA were amplified using respective PCR primers and as well as primers specific to the spike-in PCR products with KAPA Uracil+ PCR mix (Roche). The amplified products were pooled and libraries were prepared using the NEB Ultra II library preparation kit (NEB) according the manufacturer.
Bisulfite-seq, paired-end 250 with Miseq
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina MiSeq |
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| Description |
OX-BS-Wt-72_S7_L001
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| Data processing |
OxBS-seq reads were mapped to both the mouse genome mm10 GRCm38 (Dec. 2011) from UCSC and the phage Lambda genome (GenBank: J02459.1) using bsmap-2.90 (" -v 15 -w 3 -p 4 -S 1921 -q 20 -A AGATCGGAAGAGC -r 0 -R -V 2 ").
The mapping results were separated into reads belonging to the mm10 genome and each of the three loci from lambda used for oxidation and conversion efficiency calculation.
Methylation calls from lambda- and mm10-derived reads were obtained using bsmap-2.90 function methratio.py (" -u -p -g -i "correct" -x CG,CHG,CHH ").
To calculate conversion efficiencies as well as posterior probabilities of methylation, hydroxymethylation and unmodified Cytosine we used luxGLM v.0.666 (prior probabilities used for for C, hmC and mC "998,1,1", "6,2,72" and "1,998,1" respectively)
Genome_build: mouse genome mm10 GRCm38 (Dec. 2011) from UCSC
Supplementary_files_format_and_content: Posterior probabilities for C, hmC and mC for each timepoint (00h/72h) for each condition (WT/DKO) on each of the 18 queried cytosine
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| Submission date |
Jun 25, 2018 |
| Last update date |
Apr 29, 2019 |
| Contact name |
Anjana Rao |
| E-mail(s) |
arao@lji.org
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| Organization name |
La Jolla Institute for Allergy and Immunology
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| Department |
Signaling and Gene Expression
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| Lab |
Anjana Rao
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| Street address |
9420 Athena Cir
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| City |
La Jolla |
| State/province |
California |
| ZIP/Postal code |
92037 |
| Country |
USA |
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| Platform ID |
GPL16417 |
| Series (2) |
| GSE116202 |
TET enzymes augment AID expression via 5hmC modifications at the Aicda superenhancer [BS-seq] |
| GSE116208 |
TET enzymes augment AID expression via 5hmC modifications at the Aicda superenhancer |
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| Relations |
| BioSample |
SAMN09479155 |
| SRA |
SRX4290547 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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