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| Status |
Public on Dec 08, 2020 |
| Title |
METTL3_KO_SETDB1_ChIP_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
treatment: none
antibody: SETDB1 (Proteintech, #11231-1-AP)
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| Treatment protocol |
mESCs were treated with 1μM Flavopiridol, or 1μM Triptolide directly to the culture media and incubating cells with the drug for 12hrs at 37ºC, or 10μM α-Amanitin for 24 hours at 37ºC.
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| Growth protocol |
E14Tg2a murine embryonic stem cells (mES) were cultured in Dulbecco’ s Modified Eagle’ s Medium (DMEM) supplemented with 10% fetal bovine serum (FBSGibco #16000-044), 1% MEM non-essential amino acid (Gibco #11140), 55 mM β-Mercaptoethanol (Gibco#21985-023), 100 U/mL Penicillin/Streptomycin (Hyclone #SV30010), 1000 units/mL LIF (Millipore #ESG1107,) and MEK inhibitor PD0325901 (1μM) and GSK3β inhibitor CH99021 (3μM) at 37℃ with 5% CO2. For EB differentiation, embryoid bodies (EBs) were allowed to form in the absence of LIF by hanging drops containing ~1,000 mES cells/drop on petri dish lids for 2days, and then collected and transferred to standard mES culture (without LIF and MEK and GSK3β inhibitor) in non-coated petri dishes 5days.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin samples were incubated with specific antibodies in the ChIP Lysis buffer (20 mM Tris-HCl pH8.1, 150 mM NaCl, 2 mM EDTA, 1% TritonX-100 and 0.05% SDS) overnight at 4℃. The protein-DNA complexes were immobilized on pre-washed protein A/G beads (20μl per reaction). The bound fractions were washed 3 times with the Lysis buffer, and twice with the Low Salt Wash buffer (10 mM Tris-HCl, 250 mM LiCl, 1 mM EDTA, 0.5% NP-40, 0.5% Na-deoxylcholate), and once with 10 mM Tris-HCl pH8.0. Elution and reverse crosslinking were carried out in the Elution buffer (50 mM Tris-HCl pH8.0, and 1% SDS) at 65℃ for 5 hours. After 1 hour of RNase A (1unit/μl) at 37℃ and Proteinase K (1unit/μl) digestion at 55℃, DNA samples were then purified using PCR extraction kit (QIAGEN #28006).
The precipitated DNA samples were prepared for DNA deep sequencing according to manufacturer’s guidelines (SWIFT, #21096).
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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 |
Raw reads were aligned to the mm10 genome using Bowtie2 (v 2.2.5) with parameter “-k 1” to keep only one locus when a read mapped to multiple genomic regions.
PCR duplicates were removed using samtools (v1.7) rmdup.
Genome coverage bigwig files were generated by deeptools (v 3.0.2) bamCoverage with the parameter “--normalizeUsing RPKM --binSize 5”.
Genome_build: mm10
Supplementary_files_format_and_content: Genome coverage bigwig files
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| Submission date |
Feb 07, 2019 |
| Last update date |
Dec 10, 2020 |
| Contact name |
Yang Shi |
| E-mail(s) |
yshi@hms.harvard.edu
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| Organization name |
Fudan university
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| Street address |
NO. 138, Yi-Xue-Yuan Road
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| City |
Shanghai |
| ZIP/Postal code |
200032 |
| Country |
China |
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| Platform ID |
GPL21273 |
| Series (2) |
| GSE126238 |
RNA m6A modification mediated by METTL3 is important for IAP heterochromatin integrity in mESCs (ChIP-seq) |
| GSE126243 |
RNA m6A modification mediated by METTL3 is important for IAP heterochromatin integrity in mESCs |
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| Relations |
| BioSample |
SAMN10884514 |
| SRA |
SRX5347751 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3594124_METTL3_KO_SETDB1_ChIP_rep1.bw |
198.6 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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