 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on May 17, 2023 |
| Title |
FBS, ChIP Mettl3 IP, rep2 |
| Sample type |
SRA |
| |
|
| Source name |
embryonic stem cells
|
| Organism |
Mus musculus |
| Characteristics |
tissue: embryonic stem cells
cell line: E14
genotype: WT
treatment: control (FBS)
chip antibody: Mettl3 (Proteintech, 15073-1-AP)
|
| Treatment protocol |
Paused cells were treated with 200nM INK128 as mTor inhibitor for 2 weeks.
|
| Growth protocol |
ES cells were grown on gelatin for 2 weeks in FBS/LIF media, under control or paused condition.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
ESCs were spiked with 2% of human cells (HeLa), then cross-linked in 1% formaldehyde/PBS for 10min at room temperature. After quenching with 125mM glycine for 5min at room temperature, followed by 15min at 4°C, cells were washed in cold PBS and stored at −80°C. Cells were diluted at 5 million cells per 100μl in shearing buffer (1% SDS, 10mM EDTA, 50mM Tris-HCl pH8.0, 5mM NaF, Halt™ Protease Inhibitor Cocktail (Thermo Fisher), 1mM PMSF), rotating at 4°C for 30min, then 100μl lysate was passed into a microTUBE AFA Fiber Snap-Cap (Covaris). Chromatin was sheared to 200–500 bp fragments on a Covaris E220 with settings PIP 175, Duty 10%, CPB 200, for 7 minutes. Supernantants were pooled by condition, and immunoprecipitation was performed overnight using 200μl of lysate (~chromatin from 10 million cells) and 5µg of antibody (Proteintech 15073-1-AP), following the iDeal ChIP-seq kit for Transcription Factors (Diagenode) protocol. Elution, de-crosslinking, and DNA purification were performed per manufacturer’s instructions.
Libraries were constructed from ~2ng DNA and prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB).
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
FBS2
|
| Data processing |
Reads that passed quality control were trimmed of adaptors using Trim Galore! v0.4.0 and aligned to mm10 using bowtie2 v2.2.5131. Unmapped reads were then mapped to hg19. SAM files were converted to BAM files, sorted, and indexed with samtools v1.9. Bam files were deduplicated using MarkDuplicates (picard v2.18.14). Peaks were called with MACS2 (using IP samples and their input as controls) with the options --gsize 3.0e9 -q 0.05 --nomodel –broad, and annotated by intersecting center positions with RefSeq annotations. The most upstream and downstream annotated TSS and TES, respectively, were considered for each gene. Then, peak analysis was performed using DiffBind v3.0.15 with score=DBA_SCORE_TMM_READS_FULL_CPM. Cell number normalization was done with edgeR (v3.32.1), using the ratio of mm10/hg19 reads for normalization.
Assembly: mm10
Supplementary files format and content: tab-delimited text file with expression levels (log2) for Mettl3 peaks, with cell number normalization.
|
| |
|
| Submission date |
May 12, 2022 |
| Last update date |
May 17, 2023 |
| Contact name |
Evelyne Anne Collignon |
| Organization name |
Lunenfeld-Tanenbaum Research Institute
|
| Lab |
Ramalho-Santos lab
|
| Street address |
60 Murray Street, Box42,
|
| City |
Toronto |
| State/province |
ON |
| ZIP/Postal code |
M5T 3L9 |
| Country |
Canada |
| |
|
| Platform ID |
GPL19057 |
| Series (2) |
| GSE202847 |
m6A RNA methylation orchestrates transcriptional dormancy during paused pluripotency [ChIP-seq] |
| GSE202848 |
m6A RNA methylation orchestrates transcriptional dormancy during paused pluripotency |
|
| Relations |
| BioSample |
SAMN28204823 |
| SRA |
SRX15242293 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
|
|
|
|
 |
