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| Status |
Public on Jul 18, 2018 |
| Title |
OSK over expression reprogramming, day 4 |
| Sample type |
SRA |
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| Source name |
Mbd3f/- cell line, day 4 of reprogramming
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| Organism |
Mus musculus |
| Characteristics |
cell line: Mbd3f/- cell line
stage of reprogramming: day 4
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| Treatment protocol |
For secondary Mbd3f/- OSK2nd production, primary MEFs from Mbd3flox/- chimeric mice were infected with FUW-TetO-STEMCCA-OKS-mCherry and FUW-M2rtTA. IPS cells were isolated and injected into BDF2 blastocysts for the isolation of secondary MEFs. Secondary MEFs were transfected at day -3 and again at day 0 (starting reprogramming by adding DOX) with siRNA for cMyc, lMyc, nMyc or control (Stealth siRNA- mix of 3 as indicated in the paper) with RNAiMAX (Invitrogen). For molecular analysis, cells were collected at day 3 and day 7 as indicated.
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| Growth protocol |
Maintenance and reprogramming (Day 3 and beyond) of murine naïve pluripotent cells were conducted in bovine serum-free N2B27-based media or KSR media: N2B27- based media: 500ml KO-DMEM (Invitrogen), 5ml N2 supplement (Invitrogen; 17502048), 5ml B27 supplement (Invitrogen; 17504044), 15% knockout serum replacement (Invitrogen – 10828), 1mM glutamine (Invitrogen), 1% nonessential amino acids (Invitrogen), 0.1mM β-mercaptoethanol (Sigma), penicillin-streptomycin (Invitrogen), 5mg/ml BSA (Sigma). KSR media: 500 ml DMEM (Invitrogen) , 1mM glutamine (Invitrogen), 1% nonessential amino acids (Invitrogen), 0.1mM β-mercaptoethanol (Sigma), penicillin- streptomycin (Invitrogen). Naïve conditions for murine PSCs included 10μg recombinant human LIF (Millipore; LIF1005). Where indicated 2i was added 48 hours after OSKM induction: small-molecule inhibitors CHIR99021 (CH, 3μM- Axon Medchem) and PD0325901 (PD 0.2 or 1μM - Axon Medchem).
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA was isolated from indicated cell lines, and extracted from Trizol pellets by Direct-zol RNA MiniPrep kit (Zymo) according to manufacturer’s manual.
RNA was extracted from Trizol pellets, and utilized for RNA-Seq by TruSeq RNA Sample Preparation Kit v2 (Illumina) according to manufacturer’s instruction.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Myc_pert.fpkm.txt.gz
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| Data processing |
We used Illumina CASAVA 1.8.2 software to generate fastq files.
Tophat software version 2.0.10 was used to align reads to mouse mm10 reference genome
FPKM values were calculated over all genes in mm10 assembly GTF (UCSC, December 2011), using cufflinks version 2.2.1.
FC was calculated in compare to MEF siControl, replicate 1.
Genome_build: mm10
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| Submission date |
Jul 17, 2018 |
| Last update date |
Jul 18, 2018 |
| 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 |
GPL19057 |
| Series (2) |
| GSE102348 |
High-Resolution Dissection of Conducive Somatic Cell Reprogramming to Naïve Ground State Pluripotency in Mbd3/NuRD and Gatad2a/NuRD Depleted Systems [RNA-Seq] |
| GSE102518 |
High-Resolution Dissection of Conducive Somatic Cell Reprogramming to Naïve Ground State Pluripotency in Mbd3/NuRD and Gatad2a/NuRD Depleted Systems |
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| Relations |
| BioSample |
SAMN09684335 |
| SRA |
SRX4401526 |
| 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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