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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jan 24, 2021 |
| Title |
Mettl3.KO.H3K9me3.rep1 |
| Sample type |
SRA |
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| Source name |
embryonic stem cells
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| Organism |
Mus musculus |
| Characteristics |
cell type: embryonic stem cells
genotype: Mettl3.KO
chip antibody: H3K9me3 (Abcam, ab8898)
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| Growth protocol |
Male mouse embryonic stem cells (mESCs) were derived from 3.5 d.p.c inner cell mass (ICM) from female 129 mice (Stock No.: 217, Beijing Vital River Laboratory Animal Technology Co., Ltd.). Animal care and experimental protocols were approved by the animal ethics committee of the Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences. ESCs with genetic engineering, including Ythdc1-conditional knockout (cKO), MERVL::tdTomato reporter, and Halo- Ythdc1 knockin (KI) , and TurboID overexpression (OE) were constructed and cultured in feeder- free condition, using serum/LIF medium containing high-glucose Dulbecco’s modified Eagle’s medium (DMEM, HyClone, #SH30022.01), 15% fetal bovine serum (FBS, Front Biomedical, # OPT500), 1× GlutaMAX, 1× nonessential amino acids (NEAA, Gibco, #11140076), 1× Penicillin- Streptomycin (Hyclone, #SV30010), 1× Sodium Pyruvate (Gibco, #11360070), 1 mM 2- Mercaptoethanol (Gibco, # 21985023), and 1000 U/ml LIF. HEK293T (ATCC, # CRL-1126) and NIH3T3 (ATCC, #CRL-1658) cells were maintained in high-glucose DMEM supplemented with 10% FBS (Natocor, #SFBE), 1× GlutaMAX (Gibco, #35050079), and 1× NEAA. All the cell lines were cultivated at 37°C in a humidified atmosphere containing 5% CO2.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Native ChIP-seq was performed as described44. In brief, 1 × 107 cells were harvested and resuspended in 500 μl Buffer 1 (0.32 M sucrose, 15 mM Tris-HCl pH 7.5, 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 0.5 mM DTT, 0.1 mM EGTA, 0.1 mM PMSF, 1:1000 EDTA-free protease inhibitor cocktail [Roche, #4693132001]) supplemented with 0.1% IGEPAL CA-630 (Sigma, # I8896) on ice for 10 min. The lysates was carefully layered on the top of 1 ml Buffer 3 (1.2 M sucrose, 15 mM Tris-HCl pH 7.5, 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 0.5 mM DTT, 0.1 mM EGTA, 0.1 mM PMSF, 1:1000 PIC). After centrifugation (10,000 ×g for 20 min at 4°C), the nuclei were resuspended in 500 μl Buffer A (0.34 M sucrose, 60 mM KCl, 15 mM HEPES pH7.4, 15 mM NaCl, 4 mM MgCl2, 1 mM DTT, 0.1 mM PMSF, 1:1000 PIC) supplemented with 3 mM CaCl2 and 2 U/μl MNase (NEB, # M0247S) and digested for 15 min at 37°C. Then 5 mM EGTA was added to stop the reaction. After centrifuged at 13,500 ×g for 10 min at 4°C, chromatin was resuspended in 10 mM EDTA (pH 8.0) supplemented with 1 mM PMSF and 1:1000 PIC, followed by rotation at 4 °C for 2-4 h. The mixture was adjusted to 500 mM NaCl and rotated for additional 45 min. After centrifugation, the supernatant containing chromatin was diluted to 100 ng/μl with buffer B (20 mM Tris-HCl pH8.0, 5 mM EDTA, 500 mM NaCl, 0.2% Tween 20) and the fragmentation of chromatin was detected by agarose electrophoresis. A total of 2.5 μg fragmented chromatin was kept as the input. For immunoprecipitation, the reaction mixture, including 20 μg fragmented chromatin, 2 μg anti-H3K9me3 antibody (Abcam, #ab8898), and 50 μl protein A/G dynabeads (GE Healthcare) was incubated overnight with gentle rotation at 4°C. The beads were washed three times with Buffer B, and once with Buffer B without Tween 20 at 4°C for 5 min. The DNA was eluted with 300 μl elution buffer (20 mM Tris-HCl pH8.0, 20 mM EDTA, 0.5% SDS), digested with PK enzyme (NEB, #P8107S), and purified using a MinElute kit (Qiagen, #28006). The ChIP DNA library was constructed by a KAPA HTP/LTP Library Preparation Kit according to the manufacturer’s instructions.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
HiSeq X Ten |
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| Data processing |
bcl2fastq conversion software v2.17 used for basecalling.
ChIP-seq reads were aligned to the mouse mm10 genome using bowtie247 with the options ‘-p 20– very-sensitive–end-to-end–no-unal–no-mixed -X 2000’. The multimapped reads were kept for TE analysis, but only the best alignment is reported for those reads, if more than one equivalent best alignment was found, then one random alignment was reported. Whereas the reads mapping to mitochondrial DNA or unassigned sequences were discarded
Genome_build: mm10
Supplementary_files_format_and_content: bigwig
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| Submission date |
Jul 13, 2020 |
| Last update date |
Jan 24, 2021 |
| Contact name |
Jiangping He |
| E-mail(s) |
he_jiangping@grmh-gdl.cn
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| Organization name |
Guangzhou Institutes of Biomedicine and Health (GIBH), CAS
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| Street address |
Kai yuan avenue 190
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| City |
GuangZhou |
| ZIP/Postal code |
510530 |
| Country |
China |
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| Platform ID |
GPL21273 |
| Series (2) |
| GSE146464 |
High throughput sequencing for YTHDC1 binding RNAs [ChIP-seq] |
| GSE146467 |
The RNA m6A reader YTHDC1 silences retrotransposons and guards ES cell identity |
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| Relations |
| BioSample |
SAMN15518451 |
| SRA |
SRX8722027 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4669035_20200528.Mettl3.KO.10.H3K9me3.ChIP.bw |
277.7 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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