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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jul 02, 2024 |
| Title |
mESC_MeRIPseq.Dox_rep1_IP |
| Sample type |
SRA |
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| Source name |
embryo
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| Organism |
Mus musculus |
| Characteristics |
tissue: embryo
cell line: E14
cell type: mouse embryonic stem cell
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| Treatment protocol |
The mouse embryonic cells were treated with Dox or DMSO for 24 h
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| Growth protocol |
The mouse embryonic cells were cultured on 0.2% gelatin-coated dishes in Knock-out DMEM, 15% KnockOut Serum Replacement, 1% non-essential amino acids, 1% Glutamax, 1% penicillin/streptomycin, 0.1 mM β-mercaptoethanol, and 1000 U/ml Recombinant Mouse leukemia inhibitory factor Protein.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA was extracted and purified using Trizol reagent(Invitrogen) following the manufacturer's procedure. Poly(A) RNA was subsequently isolated from the total RNA using oligo dT beads (New England BioLabs, S1419S) to remove rRNA.The mRNA was then chemically fragmented into 150 nt fragments.Enrichment of m6A-modified mRNA was carried out using anti-m6A antibody (abcam, ab151230).
The IP RNA was reverse-transcribed to cDNA by SuperScript™ II Reverse Transcriptase (Invitrogen, cat. 1896649, USA), which was next used to synthesise U-labeled second-stranded DNAs with E. coli DNA polymerase I (NEB, cat.m0209, USA), RNase H (NEB, cat.m0297, USA) and dUTP Solution (Thermo Fisher, cat.R0133, USA).An A-base was added to the blunt ends of each strand, preparing for ligation to the indexed adapters. Dual-index adapters were ligated to the fragments, and size selection was performed with AMPureXP beads. After the heat-labile UDG enzyme (NEB, cat.m0280, USA) treatment of the U-labeled second-stranded DNAs, the ligated products were amplified with PCR by the following conditions: initial denaturation at 95℃ for 3 min; 8 cycles of denaturation at 98℃ for 15 sec, annealing at 60℃ for 15 sec, and extension at 72℃ for 30 sec; and then final extension at 72℃ for 5 min. At last, we performed paired-end sequencing (PE150) on an Illumina Novaseq™ 6000 platform(LC-Bio Technology CO., Ltd., Hangzhou, China) following the vendor’s recommended protocol.
MeRIP-seq
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
To get high quality clean reads, reads were further filtered by fastp (https://github.com/OpenGene/fastp, version:fastp-0.19.4). We aligned reads of all samples to the mm10 reference genome using HISAT2 (https://daehwankimlab.github.io/hisat2/, version:hisat2-2.2.1) package, and then maps the reads to the reference genome. the R package of Exomepeak(http://www.bioconductor.org/packages/2.13/bioc/html/exomePeak.html) was used to predict the site of m6A. And Called called peaks were annotated by intersection with gene architecture using R package ChIPseeker (https://bioconductor.org/packages/ChIPseeker).
Assembly: mm10
Supplementary files format and content: .bed files include methylation level
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| Submission date |
Jun 26, 2024 |
| Last update date |
Jul 02, 2024 |
| Contact name |
xiang wu |
| E-mail(s) |
3121030148@stu.cpu.edu.cn
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| Organization name |
China Pharmaceutical University
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| Lab |
Liulab
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| Street address |
639 longmiandadao
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| City |
Nanjing |
| ZIP/Postal code |
21009 |
| Country |
China |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE270881 |
Ythdf2 Suppresses the 2C-like State in Mouse Embryonic Stem Cells via Cooperation with Cnot1 [MeRIP-seq] |
| GSE270884 |
Ythdf2 Suppresses the 2C-like State in Mouse Embryonic Stem Cells via Cooperation with Cnot1 |
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| Relations |
| BioSample |
SAMN42088532 |
| SRA |
SRX25089840 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
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