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Sample GSM4664568 Query DataSets for GSM4664568
Status Public on Dec 08, 2020
Sample type SRA
Source name Embryonic stem cells
Organism Mus musculus
Characteristics cell type: Embryonic stem cells
genotype: Parental
treatment: siVIRILIZER
antibody: METTL3 (Bethyl, #A301-567A)
Treatment protocol For knockdown, mES cells were seeded in a 6-well plate, and transfected with RNAiMAX (Invitrogen, 13778-150) according to the manual. Cells were harvested after three days.
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.
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).
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model HiSeq X Ten
Data processing Raw reads were aligned to the mm10 genome using Bowtie2 (v 2.2.5).
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
Submission date Jul 09, 2020
Last update date Dec 08, 2020
Contact name Yang Shi
Organization name Fudan university
Street address NO. 138, Yi-Xue-Yuan Road
City Shanghai
ZIP/Postal code 200032
Country China
Platform ID GPL21273
Series (2)
GSE126243 RNA m6A modification mediated by METTL3 is important for IAP heterochromatin integrity in mESCs
GSE154135 RNA m6A modification mediated by METTL3 is important for IAP heterochromatin integrity in mESCs (ChIP-Seq 2)
BioSample SAMN15497020
SRA SRX8701294

Supplementary file Size Download File type/resource 128.2 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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