|
| Status |
Public on Nov 02, 2014 |
| Title |
m6A-seq in EBs, replicate C: Input |
| Sample type |
SRA |
| |
|
| Source name |
BVSC Embryoid Bodies
|
| Organism |
Mus musculus |
| Characteristics |
antibody: Input
cell type: BVSC Embryoid Bodies
strain: C57BL/6
|
| Growth protocol |
Maintenance of grounds state naïve murine pluripotent cells was conducted in FBS free N2B27-based media, and included 10μg recombinant human LIF and small-molecule inhibitors CHIR99021 (CH, 1-3 μM- Axon Medchem) and PD0325901 (PD, 1 μM - TOCRIS) termed 2i. For embryoied bodies formation 5x10^6 ESCs were disaggregated with trypsin and transfer to low non-adherent palte and culture in MEF medium (DMEM supplemented with 1% L-Glutamine, 1% Non-essential amino acids, 1% penicillin/streptomycin and 15% FBS) for 8-21 days. MEF generation: E13.5 mouse embryo was decapitated and disaggregated with trypsin and culture in MEFs medium until MEFs population were expended.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were harvested and RNA was extracted using the 5 PRIME PerfectPure RNA Cell culture Kit (cat#2302340 ), according to manufacturer's manual
Libraries were prepared according to NEB's instructions accompanying the NEBNext® Ultra™ RNA Library Prep Kit for Illumina (Cat# 7530).
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
We used Illumina CASAVA 1.8.2 software to generate fastq files.
We aligned reads to mm10 reference genome (downloaded from ensemble) using TopHat version 2.0.10. Flags used: -M
We identified enriched intervals using MACS version 1.4.1, while using the input as control in order to define a background model.
Final peak list includes peaks that appaer in at least 2 out of 3 replicates (or 2 out of 2 in MEF), their MACS fold-change is >2, and FDR < 5%, and they fall in annotated gene whose expression level was equal or over the first quartile of the expression level of all genes.
Genome_build: mm10
Supplementary_files_format_and_content: final_peaks.xlsx: final list of peaks in ESC, EBs and MEFs, and their mapping to genes
|
| |
|
| Submission date |
Oct 02, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Noa Novershtern |
| E-mail(s) |
noa.novershtern@weizmann.ac.il
|
| Organization name |
Weizmann Institute of Science
|
| Department |
Molecular Genetics
|
| Street address |
Weizmann Institute
|
| City |
Rehovot |
| ZIP/Postal code |
7610001 |
| Country |
Israel |
| |
|
| Platform ID |
GPL17021 |
| Series (2) |
| GSE61995 |
m6A mRNA Methylation Facilitates Resolution of Naïve Pluripotency Towards Differentiation (m6a-seq) |
| GSE61998 |
m6A mRNA Methylation Facilitates Resolution of Naïve Pluripotency Towards Differentiation |
|
| Relations |
| BioSample |
SAMN03092716 |
| SRA |
SRX719363 |
