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| Status |
Public on Sep 07, 2015 |
| Title |
KO1_AI |
| Sample type |
SRA |
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| Source name |
mouse embryonic stem cells
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6-ICR
cell line: mES
treatment: azido 1,3-indandione (AI) and click chemistry labeled input gDNA
pulldown: control
genotype/variation: Tdg -/-
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| Treatment protocol |
Purified mESCs genomic DNAs were fragmented to 100-400bp with NEB dsDNA fragmentas before applied to fC-CET procedures.
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| Growth protocol |
mES cell lines (Tdg -/-, and Tdg fl/fl) were cultured in feeder-free conditions, with 15% FBS, 2mM L-glutamine, 0.1 mM 2-mercaptoethanol, 1x non-essential amino acids, 1,000 units/mL LIF and 1x pen/strp.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Fragmented gDNA samples was labeled in a suspension of azido 1,3-indandione (AI) in 100 mM MES buffer (pH 6.0) for 24 h at 37°C, leading to selective and efficient labeling of 5fC. Click chemistry labeling with DBCO-S-S-Biotin (400 mM) was performed for 2 h at 37°C, followed by purification with QIAGEN PCR purification kit.The Dynabeads MyOne Streptavidin C1 (Invitrogen) was used to pull-down the biotin-labelled DNA with minor modifications on the suggested immobilizing procedure for nucleic acids. In brief, a NaOH-solution washing step (incubated in freshly prepared 0.15 M NaOH at room temperature for 10 min) was added following the canonical washing steps (5 times with 1× binding and washing buffer with 0.1% Tween-20). After that, beads were resuspended and incubated in freshly prepared 50 mM DTT to release the 5fC-containing strand for 2 h at 37 °C. Then the supernatant containing the desired DNA was purified with Micro Bio-Spin P-6 Gel Columns (Bio-Rad) to remove DTT.
The fC-CET enriched gDNAs were used directly for library preparation using the TELP protocol. TDT (NEB) enzyme was used to add a poly-dC to allow primer extension of complemwntary strand. MightyAmp DNA Polymerase (TAKARA) was used for one round of on-bead primer extension before PCR amplification. The adaptor-ligated samples were then PCR amplified using NEBNext 2× PCR Master Mix (NEB) and indexed primers (NEB). Libraries were checked using the Agilent 2100 Bioanalyzer DNA 1000 Chip. Two biological replicates of each mESCs were prepared and sequenced, which means that in parallel, two non-enriched input DNAs(Input: preAI), two AI labeled samples (Input: AI) and two pull-down output samples were sequenced simultaneously following the same procedure.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Raw reads were firstly trimmed for Poly C sequence at 3’ end. Reads shorter than 60bp were discarded. Processed reads were then mapped to the mouse genome (mm9) by bismark v0.8.3, using options -n 1 -l 40 -chunkmbs 512.
The 5fC enriched regions in each output sample were detected by using the model-based analysis of ChIP-seq (MACS) peak-calling algorithm, with the corresponding input AI sample serving as the input control. The numbers of converted (NT) and unconverted cytosines (NC) were further extracted from each output dataset. CpGs with less than 10 reads or 2 converted reads were discarded.
To detect modified CpGs that significantly enriched for 5fC, we used the binomial distribution with parameter N as (NT+ NC) and p as 5fC non-conversion rate (evaluated by spike-in sequence, p = 1.87% & 1.41% for WT replicates, p = 2.01% & 1.42% for KO replicates) to calculate the probability of observing NT or greater C-T conversion by chance. P values were further adjusted by Holm–Bonferroni method. The CpGs with adjusted p.value <0.05 in both replicates samples and located within 5fC enriched region were considered as detected 5fC sites.
Genome_build: mm9
Supplementary_files_format_and_content: BedGraph files for each dataset was generated by HOMER software. The 5fC sites detected in both wild type(WT) and TDG KO cell lines are also provided.
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| Submission date |
Feb 20, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Dali Han |
| E-mail(s) |
handali294@gmail.com
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| Organization name |
University of Chicago
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| Department |
Department of Chemisty
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| Street address |
5801 South Ellis Avenue
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| City |
Chicago |
| State/province |
IL |
| ZIP/Postal code |
60637 |
| Country |
USA |
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| Platform ID |
GPL17021 |
| Series (1) |
| GSE66144 |
Bisulfite-free and Base-resolution Analysis of 5-formylcytosine at Whole-genome Scale |
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| Relations |
| BioSample |
SAMN03360268 |
| SRA |
SRX884580 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1615104_KO1_AI.fastq.trimmed_bismark.sam.ucsc.bedGraph.gz |
43.7 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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