|
| Status |
Public on Dec 04, 2018 |
| Title |
Mettl3KO_IP_rep1 |
| Sample type |
SRA |
| |
|
| Source name |
ES cells
|
| Organism |
Mus musculus |
| Characteristics |
background: Rosa26:CRE, Bir ligase
cell type: ES cells
genotype: Mettl3KO
rip antibody: anti-m6A (Synaptic Systems; polyclonal Rabbit , Cat. No. 202 003)
|
| Growth protocol |
All samples: Mouse embryonic stem cells were cultured on gelatin-coated dishes in mES medium (DMEM (Gibco 21969-035), supplemented with 15% fetal bovine serum (Gibco), 1x non-essential amino acids (Gibco), 1mM sodium pyruvate (Gibco), 2 mM L-glutamine (Gibco), 0.1mM 2-mercaptoethanol (Sigma), 50mg/ml penicillin, 80mg/ml streptomycin and LIF conditioned medium) at 37°C in 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from mESCs was isolated using Absolutely RNA Microprep Kit (Stratagene), followed by mRNA selection using double Oligo d(T)23 (NEB) purification.
Libraries were prepared with 30 ng of m6A-IPs and duplicate input samples (200ng mRNA) using NEB-Next RNA Directional Library Preparation Kit
|
| |
|
| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
Reads were aligned to the mm10 mouse genome assembly using Bowtie with the parameters ‘-m 1 --best --strata’. SAM files were converted to BAM format, sorted and indexed using SAMtools.
MACS2 was used to call peaks of m6A enrichment for WT IP vs input samples, using the default parameters.
BigWig files for each sample were created using the qExportWig function from the QuasR package in R (Gaidatzis et al. 2014) with 50-bp bins.
Genome_build: mm10
Supplementary_files_format_and_content: bigWig: scores represent normalized counts in 50-bp bins. narrowPeak: BED6+4 format representing peak locations. Columns 5-10 are defined by MACS2 (https://github.com/taoliu/MACS).
|
| |
|
| Submission date |
Nov 07, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Jean-Yves Roignant |
| Organization name |
Institute of molecular Biology
|
| Lab |
Roignant
|
| Street address |
Ackermannweg 4
|
| City |
Mainz |
| ZIP/Postal code |
55128 |
| Country |
Germany |
| |
|
| Platform ID |
GPL17021 |
| Series (2) |
| GSE106613 |
Zc3h13/Flacc is required for adenosine methylation by bridging the mRNA binding factor Rbm15/Spenito to other components of the m6A machinery |
| GSE106614 |
Zc3h13/Flacc is required for adenosine methylation by bridging the mRNA binding factor Rbm15/Spenito to other components of the m6A machinery |
|
| Relations |
| BioSample |
SAMN07987887 |
| SRA |
SRX3369941 |
