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Sample GSM3594081 Query DataSets for GSM3594081
Status Public on Dec 08, 2020
Title rescue_WT_METTL3_ChIP_rep1
Sample type SRA
 
Source name Embryonic stem cells
Organism Mus musculus
Characteristics cell type: Embryonic stem cells
genotype: METTL3 KO+WT METTL3
treatment: none
antibody: METTL3 (Bethyl, #A301-567A)
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.
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) 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
 
Submission date Feb 07, 2019
Last update date Dec 10, 2020
Contact name Yang Shi
E-mail(s) yshi@hms.harvard.edu
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)
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
Relations
BioSample SAMN10884425
SRA SRX5347708

Supplementary file Size Download File type/resource
GSM3594081_rescue_WT_METTL3_ChIP_rep1.bw 250.7 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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