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| Status |
Public on May 17, 2023 |
| Title |
Paused, MeRIP input, rep1 |
| Sample type |
SRA |
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| Source name |
embryonic stem cells
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| Organism |
Mus musculus |
| Characteristics |
tissue: embryonic stem cells
cell line: E14
genotype: WT
treatment: paused
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| Treatment protocol |
Paused cells were treated with 200nM INK128 as mTor inhibitor for 2 weeks.
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| Growth protocol |
ES cells were grown on gelatin for 2 weeks in FBS/LIF media, under control or paused condition.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
mESCs were spiked with 2% of human cells (HeLa), then poly(A) RNA was extracted using the Magnetic mRNA Isolation Kit (NEB) with two rounds of enrichment. Immunoprecipitation of m6A methylated RNA (MeRIP) was done using the EpiMark® N6-Methyladenosine Enrichment Kit with 4µg spiked poly(A) RNA.
MeRIP libraries were constructed from 0.5-1ng of input or IP RNA and prepared using the SMARTR-seq RNA library prep v2 kit (TakaraBio), per manufacturer’s recommendations.
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| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
Libraries were trimmed of adaptors and quality-checked using Trim Galore! v0.4.0, and aligned to the mm10 transcriptome using TopHat2115 v2.0.13. Unmapped reads were then aligned to hg19.
For input samples, raw counts were obtained from the featureCounts function of subread (v1.5.0) with options: -t exon -g gene_id. Counts were imported into R, and analyzed by cell number normalization using the ratio of hg19/mm10 reads with edgeR (v3.32.1).
For m6A-IP samples, peaks were called with MACS2 (using IP samples and their input counterpart as controls and q<0.01). Peaks were annotated by intersecting center positions with RefSeq annotations, and manually verified. Peak analysis was performed using DiffBind v3.0.15, with the options minOverlap=2, score=DBA_SCORE_READS. MeRIP peaks were then first normalized using the ratio of hg19/mm10 reads in each sample for normalization. Then, peak levels were adjusted by dividing values by the ratio Input.sample/Input.average of the corresponding gene to consider expression changes. In the following differential analysis with edgeR, these normalized m6A levels were protected from further re-scaling by fixing the library size for all samples as lib.size = rep(10^6, 6) in the voom function.
Assembly: mm10
Supplementary files format and content: tab-delimited text file with expression levels (log2) for all m6A-RIP peaks, adjusted for gene expression and with cell number normalization.
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| Submission date |
May 12, 2022 |
| Last update date |
May 17, 2023 |
| Contact name |
Evelyne Anne Collignon |
| Organization name |
Lunenfeld-Tanenbaum Research Institute
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| Lab |
Ramalho-Santos lab
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| Street address |
60 Murray Street, Box42,
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| City |
Toronto |
| State/province |
ON |
| ZIP/Postal code |
M5T 3L9 |
| Country |
Canada |
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| Platform ID |
GPL21103 |
| Series (2) |
| GSE202846 |
m6A RNA methylation orchestrates transcriptional dormancy during paused pluripotency [MeRIP-seq] |
| GSE202848 |
m6A RNA methylation orchestrates transcriptional dormancy during paused pluripotency |
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| Relations |
| BioSample |
SAMN28204765 |
| SRA |
SRX15242299 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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