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| Status |
Public on Jun 30, 2017 |
| Title |
input_DNA_Untreated |
| Sample type |
SRA |
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| Source name |
embryonic stem cells, untreated, input
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| Organism |
Mus musculus |
| Characteristics |
strain/background: 129 X MF1
cell type: Oct4-GiP mES cells
passages: p30
treatment: none
chip antibody: none
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| Treatment protocol |
Vitamin C (L-ascorbic acid 2-phosphate, Sigma, A8960) was added on day 1 after seeding at 100 μg/ml. Cells were treated for 72 hours. Medium was replaced daily.
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| Growth protocol |
Oct4-GiP ES cells were cultured on tissue culture plates coated with 0.1% gelatin in 2i medium, which consists of N2B27 base medium supplemented with the MEK inhibitor, PD0325901 (1 μM, Stemgent), the GSK3β inhibitor, CHIR99021 (3 μM, Selleck Chemicals), and with ESGRO leukemia inhibitory factor (LIF) at 1,000 U/ml (Millipore).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP protocol: ES cells were crosslinked with 1% formaldehyde for 5 minutes at room temperature. Crosslinking was quenched with 125mM glycine. Crosslinked material was sonicated on a Covaris sonicator for 12 min at duty 5%, intensity 3, and bursts 200. ChIP was performed using the Diagenode LowCell# Kit. ChIP DNA was eluted, treated with RNase and Proteinase K, and reverse-cross-linked at 65°C for 4 hrs followed by purification with QIAquick kit.
Prior to library construction, sample volumes were adjusted to 35 ul with Qiagen EB buffer. All liquid handling steps were carried out on the Bravo liquid handling platform using VWorks Automation Control Software (Agilent Automation, Santa Clara, CA, USA). Enzyme mastermix dispenses and magnetic bead clean-up steps were performed using the Bravo configured with the 96LT pipetting head and 180μl disposable tips (Agilent Technologies, catalogue number 08585-002). End Repair and 5' Phosphorylation reaction (35uL DNA sample, 5uL of 10X NEB 2 buffer, 2uL of 25mM ATP, 2uL of 10mM dNTP, 10U T4 Polynucleotide Kinase, 4.5U T4 DNA Polymerase, 1U Klenow Large Fragment DNA Polymerase, and ultrapure water to a total reaction volume of 50uL (New England Biolabs Ipswich, MA, USA) was incubated for 30 minutes at room temperature. DNA samples were then purified using house-made magnetic bead solution (1M NaCL, 23% PEG, Sera-Mag Speedbeads (Fisher Scientific, Pittsburgh PA, USA)) with final PEG concentration of 13.87%. DNA was eluted from the beads in 35uL volume with Qiagen EB buffer. To enable ligation to the adaptors, a single dA overhang was added to the 3' ends of DNA fragments (35uL DNA, 5uL of 10X NEB 2 buffer, 1uL of 10mM dATP, 5U Klenow Fragment (3'→5 ' exo–), and ultrapure water to a total reaction volume of 50uL (New England Biolabs Ipswich, MA, USA)). A-addition reaction was incubated at 37C for 30 minutes. The product was then purified using house-made magnetic bead solution (1M NaCL, 23% PEG, Sera-Mag Speedbeads (Fisher Scientific, Pittsburgh PA, USA)) with final PEG concentration of 13.87%. DNA was eluted from the beads in 35uL volume with Qiagen EB buffer. Next, short adaptors containing sequences required downstream in the sequencing workflow were ligated to the dA-tailed DNA fragments (35uL DNA, 12uL of 5X Quick Ligation Buffer, 2000U Quick T4 DNA Ligase, 2uL of 0.5uM Illumina short sequencing adaptor, and ultrapure water to a total volume of 60uL). Ligation reaction was performed at room temperature overnight. To remove adaptor dimers and fragments below 200bp, the ligation product was then purified two times using house-made magnetic bead solution (1M NaCL, 20% PEG, Sera-Mag Speedbeads (Fisher Scientific, Pittsburgh PA, USA)) with final PEG concentrations of 8.89% and 10.91%, respectively. Adaptor-ligated DNA fragments were eluted from the beads with 35uL of Qiagen EB buffer. Adaptor ligated libraries were PCR amplified and barcoded by custom indexing primers in a 60 ul PCR reaction (35uL DNA, 12uL of 5X High fidelity buffer, 1uL of 10mM dNTPs, 1.5uL of DMSO, 1uL of 25uM PCR primer 1.0, 2uL of 12.5uM custom indexing primer (added separately to each well), 1U Phusion Hot Start II, and ultrapure water to a total volume of 60uL (Fisher Scientific, Pittsburgh PA)). PCR amplification was carried out with the following cycling conditions: 98C for 30 seconds, 10 cycles of (15 seconds at 98C (denaturation), 30 seconds at 65C (annealing), 30 seconds 72C (extension)), followed by 5 min at 72C. PCR amplified libraries were size selected to remove primer dimers with house-made magnetic bead solution (1M NaCL, 20% PEG, Sera-Mag Speedbeads (Fisher Scientific, Pittsburgh PA, USA)) with final PEG concentration of 9.19%. Final library product was eluted from the beads with 35uL of Qiagen EB buffer. The average size was determined by running 1uL of each final library product on the Agilent HS DNA assay chip. Libraries were quantified using Qubit HS DNA assay, pooled equal-molar and quantified for sequencing with Kapa Sybr Fast Complete Universal qPCR kit using manufacturer recommendations.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Paired-end sequenced reads were aligned to mm10 genome using BWA 0.5.7 and default parameters (for both aln and sampe steps). Aligned results were reported in the BAM format. Three reads partitions were merged followed by marking duplicated reads using PICARD 1.71 with default settings.
WIG files calculated by profiling paired fragment density after collapsing duplicated fragments and using mapping quality threshold of BWA q>=5.
Genome_build: mm10 (GRCm38)
Supplementary_files_format_and_content: WIG files contain ChIP-seq data processed as described.
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| Submission date |
Jul 05, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Miguel Ramalho-Santos |
| E-mail(s) |
mrsantos@diabetes.ucsf.edu
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| Organization name |
UCSF
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| Department |
Eli and Edythe Broad Center of Regeneration Medicine
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| Lab |
Santos lab
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| Street address |
35 Medical Center Way
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| City |
San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94143 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE84009 |
Vitamin C Induces Specific Demethylation of H3K9me2 in Mouse Embryonic Stem Cells via Kdm3a/b |
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| Relations |
| BioSample |
SAMN05358032 |
| SRA |
SRX1897484 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2225282_input_none.wig.gz |
248.8 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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