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| Status |
Public on Nov 02, 2014 |
| Title |
Mettl3-KO EBs 8h after Actinomycin treatment |
| Sample type |
SRA |
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| Source name |
Embroid bodies
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| Organism |
Mus musculus |
| Characteristics |
genotype: Mettl3-KO
cell type: Embroid bodies
time after treatment: 8h
strain: C57BL/6
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| 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.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA was fragmented into average size of 300 nucleotides by incubation of the RNA in 95oC for 4:30 minutes (NEBNext Magnesium RNA Fragmentation Module). The 3’ polyadenylated fragments were enriched by selection on poly dT beads (Dynabeads Invitrogen).
Strand specific cDNA was synthesized using a poly T-VN oligo (18 T) and Affinity Script RT enzyme (Agilent). Double strand DNA was obtained using Second strand synthesis kit (NEBNext second strand synthesis module). DNA was purified by adding paramagnetic SPRI beads (Agencourt AMPure XP, Beckman Coulter) pipette-mixed 15 times and incubated for 2 minutes. Supernatant were separated from the beads using a 96-well magnet for 4 minutes. Beads were washed on the magnet with 70% ethanol and then air dried for 4 minutes. The DNA was eluted in EB buffer (10 mM Tris-HCl pH 8.0) by pipette mixing 25 times. For the remainder of the library construction process (DNA end-repair, A-base addition, adaptor ligation and enrichment) a general SPRI cleanup involves addition of buffer containing 20% PEG and 2.5 M NaCl to the DNA reaction. DNA ends are first repaired by T4 polymerase (NEBNext), next, T4 polynucleotide kinase (NEB) adds a phosphate group at the 5′ ends. An adenosine base is then added to the blunt-ended fragments, using Klenow enzyme (NEB), and a barcode Illumina compatible adaptor (IDT) was ligated to each fragment using T4 quick ligase (NEB). DNA fragments were amplified by 12 cycles of PCR (Kapa HiFi) using specific primers (IDT) to the ligated adaptors. Library quantity was determined by Qubit Fluorometric Quantitiation (Life Technologies) and the quality of each library was analyzed by Tapestation (Agilent).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
We used Illumina CASAVA 1.8.2 software to generate fastq files.
We aligned reads to bowtie mm10 index, using bowtie2, version 2.1.0
Gene expression levels were estimated in the following way: each gene was divided to 50-bp bins, from its transcription-start-site and to 3kb after its transcription-end-site (based on UCSC “refGene” table). The number of reads in each bin was counted, and the bin with the highest number of reads was chosen as the representative expression level of that gene.
These expression levels were normalized by the sample size, such that each level represents RPM.
Genome_build: mm10
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| Submission date |
Oct 02, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Noa Novershtern |
| E-mail(s) |
noa.novershtern@weizmann.ac.il
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| Organization name |
Weizmann Institute of Science
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| Department |
Molecular Genetics
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| Street address |
Weizmann Institute
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| City |
Rehovot |
| ZIP/Postal code |
7610001 |
| Country |
Israel |
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| Platform ID |
GPL17021 |
| Series (2) |
| GSE61994 |
m6A mRNA Methylation Facilitates Resolution of Naïve Pluripotency Towards Differentiation (3p-Seq) |
| GSE61998 |
m6A mRNA Methylation Facilitates Resolution of Naïve Pluripotency Towards Differentiation |
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| Relations |
| Reanalyzed by |
GSE80797 |
| BioSample |
SAMN03092705 |
| SRA |
SRX719342 |
| 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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