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| Status |
Public on Jan 17, 2020 |
| Title |
WT_Input_r1 |
| Sample type |
SRA |
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| Source name |
Embryonic stem cells
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| Organism |
Mus musculus |
| Characteristics |
cell type: mouse embryonic stem cells
strain: C57BL/6
antibody: none
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| Treatment protocol |
mESCs were resuspended in growth media with a concentration of 10e6 ml-1 and cross-linked by adding 1% formaldehyde directly to the media, slow shaked 20 min at room temperature. Cross-linking was stopped by adding glycine to a final concentration of 0.125 M and incubating for 5 min at room temperature with slowly shaking. The media was removed and the cells were washed twice with ice-cold PBS.
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| Growth protocol |
Cells were maintained in DMEM (Invitrogen) supplemented with 15% FBS, 1% nucleosides (100×),1 mM L-glutamine,1% nonessential amino acid, 0.1 mM 2-mercaptoethanol, 1,000 U/ml LIF, 3 μM CHIR99021 and 1 μM PD0325901 37 °C in 5% CO2.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
The cells were then collected in lysis buffer (0.1% SDS, 2 mM EDTA, 1 x protease inhibitors, 20 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1% Triton) and the lysates were sonicated by a Bioruptor UCD-200 (Diagenode) to result in DNA fragments of 200 to 500 bp in length. Cellular debris was removed by centrifugation and the lysates were diluted 1:10 in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 167 mM NaCl, 1 x protease inhibitors, 16.7 mM Tris–HCl, pH 8.0). Take 1% of sonicated chromatin as Input control. Washed Protein G Magnetic beads (Invitrogen) two times with ChIP buffer, eluted beads in 1 mL of ChIP buffer, added METTL3 antibody (Abcam) and incubated the mixture 2 hours on rotating platform at 4 °C. The washed the antibody-coated beads with ChIP buffer three times. Chromatin solutions were incubated overnight at 4 °C with rotation with antibodies-coated beads. The beads were washed sequentially for 3 min by rotation with 1 ml of the following buffers: low salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris–HCl, pH 8.0), high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 500 mM NaCl, 20 mM Tris–HCl, pH 8.0) and LiCl wash buffer (0.25 M LiCl, 1% Nonidet P-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris–HCl, pH 8.0). Finally, the beads were washed twice with 1 ml TE buffer (1 mM EDTA, 10 mM Tris–HCl, pH 8.0). The immuno-complexes and Input were then eluted by adding 400 μl of elution buffer (100 mM NaHCO3, 1% SDS) plus 18 μl 5 M NaCl and incubating for 4 hrs at 65 °C to reverse protein-DNA crosslinks. The remaining proteins were digested by adding 5 μl of proteinase K (Promega) and incubating overnight at 65 °C. The DNA was recovered by phenol/chloroform/isoamyl alcohol (25:24:1) extractions and precipitated with 0.1 volume of 3 M sodium acetate, pH 5.2, and 3 volumes of ethanol using glycogen as carrier.
Library preparation was performed by using KAPA HyperPlus Kits (Kapabiosystems) according to the manufacturer’s protocols.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
Basecalls performed using CASAVA
Raw reads were trimmed with Trimmomatic-0.38, and then uniquely mapped to mouse genome (mm10, version M19, 2018-08-30) and Drosophila melanogaster chromatin (Spike-in Chromatin) using bowtie (version 1.2.2) allowing one mismatch.
METTL3 peaks were called using HOMER in factor style with parameter ‘-F 1’ and ‘-L 1’ separately.
Genome_build: mm10
Supplementary_files_format_and_content: Tab-delimited peak locations together with peak summit and pvalue reported by HOMER for each sample.
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| Submission date |
Jul 01, 2019 |
| Last update date |
Jan 17, 2020 |
| Contact name |
Xiaoyang Dou |
| E-mail(s) |
[email protected]
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| Organization name |
Center for Excellence in Molecular Cell Science, CAS
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| Street address |
320 Yue Yang Road
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| City |
Shanghai |
| ZIP/Postal code |
200031 |
| Country |
China |
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| Platform ID |
GPL21103 |
| Series (2) |
| GSE133581 |
The RNA N6-methyladenosine regulates chromatin remodeling and transcription [seq_mettl3ChIP] |
| GSE133600 |
The RNA N6-methyladenosine regulates chromatin remodeling and transcription |
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| Relations |
| BioSample |
SAMN12172224 |
| SRA |
SRX6385221 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data not applicable for this record |
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