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| Status |
Public on Dec 08, 2020 |
| Title |
IAPEz_ChIRP_rep2 |
| 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: parental
treatment: none
probe: IAPEz-int DNA
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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 |
E14 ES cells were crosslinked with 3% formaldehyde for 30 min at room temperature. Crosslinking was then quenched with 0.125 M glycine for 5 min. Chromatin was then solubilized by sonicating in lysis buffer (50 mM Tris 7.0, 10 mM EDTA, 1% SDS, 0.5 mM DTT, and RNase Inhibitors). Chromatin is diluted in two times volume of hybridization buffer (750mM NaCl, 1% SDS, 50 mM Tris 7.0, 1 mM EDTA, 15% Formamide, 0.5 mM DTT, and RNase Inhibitors). 10 pmol probes were added to 3 ml of diluted chromatin, which was mixed by end-to-end rotation at 37 °C for 6 hr. Streptavidin beads were washed three times in nuclear lysis buffer and added into the reaction, which was mixed for another 45 min at 37 °C. Beads were captured by magnets (Invitrogen) and washed five times with wash buffer (2x SSC, 0.5% SDS, add DTT and PMSF fresh). chromatin was reverse crosslinked at 65 °C overnight. After 1 hour of RNase A (1unit/μl) at 37 °C and Proteinase K (1unit/μl) digestion at 55 °C, 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).
The precipitated DNA samples were prepared for DNA deep sequencing according to manufacturer’s guidelines (SWIFT, #21096).
ChIRP-Seq
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| 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).
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 |
Jul 09, 2020 |
| Last update date |
Dec 08, 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) |
| GSE126243 |
RNA m6A modification mediated by METTL3 is important for IAP heterochromatin integrity in mESCs |
| GSE154137 |
RNA m6A modification mediated by METTL3 is important for IAP heterochromatin integrity in mESCs (ChIRP-Seq) |
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| Relations |
| BioSample |
SAMN15496989 |
| SRA |
SRX8701303 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4664577_IAPEz_ChIRP_rep2.bw |
274.8 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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