Dataset Information

Dissecting the specific role of Oct4 during the early stage of reprogramming

ABSTRACT: Differentiated cells can be reprogrammed into induced pluripotent stem cells (iPSCs) following the forced overexpression of a specific combination of transcription factors, of which Oct4 is essential. To better understand the mechanism underlying iPSC reprogramming, we investigated the immediate transcriptional response to ectopic Oct4 expression in somatic cells. To this end, we derived different somatic cell types from Oct4-inducible transgenic mice and induced Oct4 expression for different time periods. Using microarray analysis, we have identified early transcriptional Oct4 targets, including genes not previously known to interact with Oct4. Comparison of the sets of Oct4-regulated genes revealed pronounced differences among the individual cell types. Furthermore, a notable up-regulation of pluripotent markers could not be detected. In contrast, we observed a down-regulation of somatic markers specific to each cellular population. This finding suggests that Oct4 interferes with the somatic transcriptional program in a cell type-specific manner, thereby promoting an unstable epigenetic state that facilitates reprogramming rather than the direct initiation of a pluripotent gene expression network. Finally, we show that Mgarp, a gene expressed in pluripotent cells, is commonly up-regulated in all cell types after Oct4 induction. Unexpectedly, Mgarp expression decreases during the first steps of reprogramming due to a Klf4-dependent inhibition. Our data show that Oct4 counteracts Klf4 repressive activity and enhances Mgarp expression at the late stages of reprogramming. This temporal pattern of Mgarp expression is crucial for the efficient generation of iPSCs. In summary, our study provides new insights into the specific role that Oct4 plays during the early stages of reprogramming. Different somatic cell types were derived from transgenic mice containing both a tetracycline-inducible transactivator (rtTA-M2) and a tetracycline operon-controlled Oct4 expression cassette (TO) (Hochedlinger 2005, Cell 121:465-477) that were mated with GOF18-GFP mice in which the green fluorescent protein (GFP) is driven by 18 kb of the Oct4 regulatory region (Oct4-GFP) (Yoshimizu 1999, Dev Growth Differ 41:675-684), thus indicating endogenous Oct4 expression. In addition, a control neural stem cell (CtrlNSC) line was generated from transgenic mice that entail a tetracycline-inducible transactivator (irtTA-VP16-GBD) (Anastassiadis 2002, Gene 298:159-172) plus an OG2 Oct4-GFP reporter transgene (Yoshimizu 1999),but lack a TO cassette. A second control consisted of wild type (C57BL/6) mouse embryonic fibroblasts (CtrlMEF). Oct4-inducible mouse embryonic fibroblasts (TO-MEFs) and neural stem cell (TO-NSCs) were derived from 14.5 days post coitum (d.p.c.) embryos and cultured as previously described (Conti 2005, PLoS Biol 3:e283). Primary bone marrow cells (TO-BMCs) were isolated from 6-week old mice after flushing femora and tibiae and plated onto dishes with a combined coating of gelatin (PAA, Pasching, Austria), laminin (Santa Cruz Biotechnology, Santa Cruz, CA), and poly-L-lysine (Sigma, St. Louis, MO). Adherent cells were then cultivated in DMEM Low Glucose (Gibco, Carlsbad, CA) with 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM/L glutamine (all PAA). The transgenic Oct4 expression was induced by either 6 µg/ml doxycycline (Sigma) or by 2 µg/ml doxycycline (Dox) plus 10–7 M dexamethasone (Sigma), depending on the respectively contained tetracycline transactivator. 5-azacytidine (10 nM, Sigma) and cycloheximide (30 µg/ml, Sigma) were applied as indicated. Generation, culture, and differentiation of ESCs and iPSCs were performed as previously described (Tiemann 2011, Cytometry A 79:426-435). Microarray Gene Expression Analysis Samples from Oct4-inducible cell types and controls were collected at different time points (0, 6, 12, and 24 hours after induction). Biotin-labeled cRNA was prepared with the linear TotalPrep RNA Amplification Kit (Ambion, Austin, TX) from 500 ng original RNA and hybridized onto MouseRef 8 v2.0 Expression BeadChips (Illumina, San Diego, CA). The chips were stained with streptavidin-Cy3 (GE Healthcare, Little Chalfont, UK) and scanned using the iScan reader (Illumina). Bead intensities were mapped to gene information using BeadStudio 3.2 (Illumina) and background correction was performed using the Affymetrix robust multiarray analysis model. Variance stabilization was performed by log2 scaling and the R-Bioconductor lumi package was used for normalization.

ORGANISM(S): Mus musculus

PROVIDER: GSE47061 | GEO | 2014/05/13

SECONDARY ACCESSION(S): PRJNA203420

REPOSITORIES: GEO

ACCESS DATA

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

Project description:Differentiated cells can be reprogrammed into induced pluripotent stem cells (iPSCs) following the forced overexpression of a specific combination of transcription factors, of which Oct4 is essential. To better understand the mechanism underlying iPSC reprogramming, we investigated the immediate transcriptional response to ectopic Oct4 expression in somatic cells. To this end, we derived different somatic cell types from Oct4-inducible transgenic mice and induced Oct4 expression for different time periods. Using microarray analysis, we have identified early transcriptional Oct4 targets, including genes not previously known to interact with Oct4. Comparison of the sets of Oct4-regulated genes revealed pronounced differences among the individual cell types. Furthermore, a notable up-regulation of pluripotent markers could not be detected. In contrast, we observed a down-regulation of somatic markers specific to each cellular population. This finding suggests that Oct4 interferes with the somatic transcriptional program in a cell type-specific manner, thereby promoting an unstable epigenetic state that facilitates reprogramming rather than the direct initiation of a pluripotent gene expression network. Finally, we show that Mgarp, a gene expressed in pluripotent cells, is commonly up-regulated in all cell types after Oct4 induction. Unexpectedly, Mgarp expression decreases during the first steps of reprogramming due to a Klf4-dependent inhibition. Our data show that Oct4 counteracts Klf4 repressive activity and enhances Mgarp expression at the late stages of reprogramming. This temporal pattern of Mgarp expression is crucial for the efficient generation of iPSCs. In summary, our study provides new insights into the specific role that Oct4 plays during the early stages of reprogramming. Different somatic cell types were derived from transgenic mice containing both a tetracycline-inducible transactivator (rtTA-M2) and a tetracycline operon-controlled Oct4 expression cassette (TO) (Hochedlinger 2005, Cell 121:465-477) that were mated with GOF18-GFP mice in which the green fluorescent protein (GFP) is driven by 18 kb of the Oct4 regulatory region (Oct4-GFP) (Yoshimizu 1999, Dev Growth Differ 41:675-684), thus indicating endogenous Oct4 expression. In addition, a control neural stem cell (CtrlNSC) line was generated from transgenic mice that entail a tetracycline-inducible transactivator (irtTA-VP16-GBD) (Anastassiadis 2002, Gene 298:159-172) plus an OG2 Oct4-GFP reporter transgene (Yoshimizu 1999),but lack a TO cassette. A second control consisted of wild type (C57BL/6) mouse embryonic fibroblasts (CtrlMEF). Oct4-inducible mouse embryonic fibroblasts (TO-MEFs) and neural stem cell (TO-NSCs) were derived from 14.5 days post coitum (d.p.c.) embryos and cultured as previously described (Conti 2005, PLoS Biol 3:e283). Primary bone marrow cells (TO-BMCs) were isolated from 6-week old mice after flushing femora and tibiae and plated onto dishes with a combined coating of gelatin (PAA, Pasching, Austria), laminin (Santa Cruz Biotechnology, Santa Cruz, CA), and poly-L-lysine (Sigma, St. Louis, MO). Adherent cells were then cultivated in DMEM Low Glucose (Gibco, Carlsbad, CA) with 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM/L glutamine (all PAA). The transgenic Oct4 expression was induced by either 6 µg/ml doxycycline (Sigma) or by 2 µg/ml doxycycline (Dox) plus 10–7 M dexamethasone (Sigma), depending on the respectively contained tetracycline transactivator. 5-azacytidine (10 nM, Sigma) and cycloheximide (30 µg/ml, Sigma) were applied as indicated. Generation, culture, and differentiation of ESCs and iPSCs were performed as previously described (Tiemann 2011, Cytometry A 79:426-435). Microarray Gene Expression Analysis Samples from Oct4-inducible cell types and controls were collected at different time points (0, 6, 12, and 24 hours after induction). Biotin-labeled cRNA was prepared with the linear TotalPrep RNA Amplification Kit (Ambion, Austin, TX) from 500 ng original RNA and hybridized onto MouseRef 8 v2.0 Expression BeadChips (Illumina, San Diego, CA). The chips were stained with streptavidin-Cy3 (GE Healthcare, Little Chalfont, UK) and scanned using the iScan reader (Illumina). Bead intensities were mapped to gene information using BeadStudio 3.2 (Illumina) and background correction was performed using the Affymetrix robust multiarray analysis model. Variance stabilization was performed by log2 scaling and the R-Bioconductor lumi package was used for normalization. 17 samples were analyzed. ESC: Mouse Embryonic Stem Cell (ESC) TO_MEF_0: Tetracycline Oct4 (TO) Mouse Embryonic Fibroblast (MEF), time 0h TO_MEF-6: Tetracycline Oct4 (TO) Mouse Embryonic Fibroblast (MEF), time 6h TO_MEF-12: Tetracycline Oct4 (TO) Mouse Embryonic Fibroblast (MEF), time 12h TO_MEF-24: Tetracycline Oct4 (TO) Mouse Embryonic Fibroblast (MEF), time 24h TO_BMC-0: Tetracycline Oct4 (TO) Mouse Bone Marrow Cell (BMC), time 0h TO_BMC-6: Tetracycline Oct4 (TO) Mouse Bone Marrow Cell (BMC), time 6h TO_BMC-12: Tetracycline Oct4 (TO) Mouse Bone Marrow Cell (BMC), time 12h TO_BMC-24: Tetracycline Oct4 (TO) Mouse Bone Marrow Cell (BMC), time 24h TO_NSC-0: Tetracycline Oct4 (TO) Mouse Neural Stem Cell (NSC), time 0h TO_NSC-6: Tetracycline Oct4 (TO) Mouse Neural Stem Cell (NSC), time 6h TO_NSC-12: Tetracycline Oct4 (TO) Mouse Neural Stem Cell (NSC), time 12h TO_NSC-24: Tetracycline Oct4 (TO) Mouse Neural Stem Cell (NSC), time 24h Ctrl_MEF_0: Control wild type Mouse Embryonic Fibroblast (MEF) untreated Ctrl_MEF_24: Control wild type Mouse Embryonic Fibroblast (MEF) 24h treated Ctrl_NSC_0: Control wild type Mouse Neural Stem Cell (NSC) untreated Ctrl_NSC_24: Control wild type Mouse Neural Stem Cell (NSC) 24h treated

Project description:Pluripotency can be induced from different cell types using different methods. Several studies have reported that iPSC retain an epigenetic memory from the tissue from which they were derived. However, the influence of the method used for reprogramming in the final pluripotent state has not been investigated yet. Therefore, we have taken advantage of germline stem cells (GSCs) that can be converted into germline pluripotent stem cells (gPSCs) using specific culture conditions or into induced pluripotent stem cells (iPSCs) after the forced expression of four transcription factors. Here, we compare the gene expression profile, imprinting methylation pattern and differentiation potential between GSC-derived iPSCs and gPSCs. Our analysis demonstrates that both reprogramming methods induce equivalent levels of pluripotency and that a donor GSC transcriptional memory is retained only at early steps after reprogramming. In summary, our results show that different reprogramming methods generate undistinguishable pluripotent states and exclude a method-dependent epigenetic memory. Cell Culture. All the cell lines used in this study were derived from OG2 mice. Unless otherwise noted, all cell culture reagents were purchased from Gibco or PAA. Neonatal GSCs derived from 10 days postpartum (d.p.p) murine testis and adult GSCs derived from 35 d.p.p murine testis were derived as previously described and cultured on irradiated C3H feeder cells in a medium composed of StemPro-34 SFM (including supplement), N2 supplement (1:100), 1 % heat-inactivated fetal calf serum (FCS), 5 mg ml-1 bovine serum albumin (BSA) fraction V, 100 U ml-1 penicillin, 100 µg ml-1 streptomycin, 2 mM L‑glutamine, 0.1 mM non-essential amino acids (NEAA), 0.1 mM β‑mercaptoethanol, MEM vitamins (1:100), 6 mg ml-1 D‑(+)‑glucose (Sigma), 30 mg ml-1 pyruvic acid, 0.1 % DL‑lactic acid (Sigma), 60 ng ml-1 progesterone (Sigma), 30 ng ml-1 β‑estradiol (Sigma), 15 ng ml-1 mouse EGF (Peprotech), 10 ng ml-1 human bFGF (Peprotech), 10 ng ml-1 human GDNF (Peprotech), and 1000 U ml-1 mouse LIF (in‑house preparation). For maintenance culture, GSCs were passaged at a 1:2‑ratio once a week after digestion with 0.05 % trypsin/EDTA. In addition, ESCs, iPSC and gPSC were cultured on irradiated C3H feeders in the following medium: Knockout DMEM, 20% Knockout Serum Replacement, penicillin, streptomycin, L‑glutamine, NEAA, β‑mercaptoethanol, and LIF. Generation of pluripotent cell lines. iPSC were generated using tetracycline-inducible lentiviruses. For the lentiviral production, 293T cells were plated at 50 % confluency in 10-cm dishes one day prior to transfection. The next day, the lentiviral vector (3 µg) together with the packaging plasmids psPAX2 (2 µg) and pMD2.G (1 µg) (Addgene plasmids #12260 and #12259, respectively) were transfected using FuGENE 6 reagent (Roche) according to the manufacturer’s protocol. After 48 h, viral-containing supernatants were filtered (0.45 µm), concentrated around 30-fold by centrifugation (26,000 × g, 2 h, 4 °C) and stored at -80 °C. For the generation of iPSCs, a single-cell suspension of 50,000 GSCs was infected overnight with five tetracycline-inducible lentiviruses (5–20 µl concentrated supernatant each) encoding for Oct4, Sox2, Klf4, c-Myc, and the reverse tetracycline-transactivator M2-rtTA (Addgene plasmids #20323, #20326, #20322, #20324, and #20342) in the presence of 4 µg ml-1 polybrene (Sigma). One week after the infection, the expression of the viral transgenes was induced by adding 4 µg ml-1 doxycycline (Sigma) to the medium. MEFs-derived iPSCs were generated using the same protocol. gPSC were generated following a previous reported protocol. Briefly, GSCs were seeded at a density of approx. 1000 cells cm-2 on feeder cells and were not splitted until gPSC colonies were observed. Both iPSC and gPSC colonies were identified by Oct4-GFP expression, manually picked and expanded under ESC conditions. In vitro differentiation of pluripotent cells. Embryoid body (EB) formation was induced in suspension culture adopting the hanging drop method (600 cells in 20 µl) for 5 days in ESC medium without LIF. EBs were seeded on gelatine-coated plates and further cultured for 14 days in the same medium. Chimera formation. Aggregations of putative pluripotent cells with denuded 8-cell stage embryos were performed as previously reported. After overnight incubation at 37 °C and 5 % CO2, 11–14 aggregates were transferred into the uterine horns of 2.5 d.p.c pseudo-pregnant surrogate mothers (mated with vasectomized males). The chimeric pups were obtained and dissected at 14.5 d.p.c. Microarray gene expression analysis. Purified cRNA samples were prepared with the linear TotalPrep RNA Amplification Kit (Ambion) from 500 ng original RNA, involving synthesis of double-stranded cDNA by T7-promoter-linked oligo(dT) primers and 14 hours of in-vitro transcription incorporating biotin-labelled nucleotides. Hybridizations onto MouseRef 8 v2.0 Expression BeadChips (Illumina) was performed according to the manufacturer’s recommendations. Two replicates were hybridized from each sample. Chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using an iScan reader (Illumina). The bead intensities were mapped to gene information using BeadStudio 3.2 (Illumina). Background correction was performed using the Affymetrix robust multiarray analysis (RMA) background correction model. Variance stabilization was performed by log2 scaling, and gene expression normalization was calculated with the method implemented in the lumi package of R-Bioconductor.

Project description:Several Sox family members, small molecules, and inhibitors of the TGF-β pathway can replace exogenous Sox2 during the generation of induced pluripotent stem cells (iPSCs), suggesting that SOX2 is dispensable for initiation of reprogramming. However, the time point at which the endogenous Sox2 locus is activated in these conditions was not thoroughly evaluated. To identify the Sox2-independent reprogramming steps, we excluded Sox2 from the reprogramming cocktail and used Sox2-deficient mouse embryonic fibroblasts. We show that reprogramming is initiated in the absence of SOX2 as shown by formation of pre-iPSC colonies that have undergone a mesenchymal-to-epithelial transition and that have activated early reprogramming markers. However, these iPSC-like intermediates cannot progress through the final stabilization phase of reprogramming in the absence of SOX2. Overall, our results provide a better understanding of the molecular events underlying the induction of pluripotency and of the role that SOX2 plays in the process. Both pLV-tetO-E1A-2A-Cherry and pLV-tetO-Cherry lentiviral constructs were cloned in house and defective-lentiviral particles were produced as previously described (Han et al. 2013). Next, 5x10 4 ESCs containing a tetracycline-inducible transactivator (irtTA-VP16-GBD, Anastassiadis et al. 2002) plus an Oct4-GFP reporter transgene (Yoshimizu et al. 1999) were transduced twice using 20 µl of replication-defective lentiviral particles plus polybrene (4 µg/ml, Sigma). Cells were washed 24 h after transduction and subsequently induced with doxycycline (1 µg/ml, Sigma) and dexamethasone (10-7 M, Sigma). Samples were harvested at several time-points, 0, 3, 6, 9, 12, 24, and 48 hours after induction, and used for microarray analysis as previously reported (Tapia et al. 2012). Cells transduced with pLV-tetO-Cherry and induced for 48 h were used as control. RNA wasprocessed using the RNeasy micro and mini kits with on-column DNase digestion of QIAGEN as per the manufacturer’s instructions. Integrity of RNA samples was quality checked using a 2100 Bioanalyzer (Agilent). When possible, 300 ng of total RNA per sample was used as starting material for linear amplification (Illumina TotalPrep RNA Amplification Kit, Ambion), which involved synthesis of T7-linked double-stranded cDNA and 14 hr of in-vitro transcription incorporating biotin-labeled nucleotides. Purified and labeled cRNA was then quality checked on a 2100 Bioanalyser and hybridized as biological or technical duplicates for 18 hr onto MouseRef-8 v2 gene expression BeadChips (Illumina), following the manufacturer's instructions. After being washed, the chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using an iScan reader and accompanying software. Bead intensities were mapped to gene information using BeadStudio 3.2 (Illumina). Background correction was performed using the Affymetrix Robust Multi-array Analysis (RMA) background correction model. Variance stabilization was performed using log2 scaling, and gene expression normalization was calculated with the quantile method implemented in the lumi package of R-Bioconductor. Data post-processing and graphics were performed with in-house developed functions in Matlab.

Project description:Pluripotency can be induced from different cell types using different methods. Several studies have reported that iPSC retain an epigenetic memory from the tissue from which they were derived. However, the influence of the method used for reprogramming in the final pluripotent state has not been investigated yet. Therefore, we have taken advantage of germline stem cells (GSCs) that can be converted into germline pluripotent stem cells (gPSCs) using specific culture conditions or into induced pluripotent stem cells (iPSCs) after the forced expression of four transcription factors. Here, we compare the gene expression profile, imprinting methylation pattern and differentiation potential between GSC-derived iPSCs and gPSCs. Our analysis demonstrates that both reprogramming methods induce equivalent levels of pluripotency and that a donor GSC transcriptional memory is retained only at early steps after reprogramming. In summary, our results show that different reprogramming methods generate undistinguishable pluripotent states and exclude a method-dependent epigenetic memory. Cell Culture. All the cell lines used in this study were derived from OG2 mice. Unless otherwise noted, all cell culture reagents were purchased from Gibco or PAA. Neonatal GSCs derived from 10 days postpartum (d.p.p) murine testis and adult GSCs derived from 35 d.p.p murine testis were derived as previously described and cultured on irradiated C3H feeder cells in a medium composed of StemPro-34 SFM (including supplement), N2 supplement (1:100), 1Â % heat-inactivated fetal calf serum (FCS), 5Â mgÂ ml-1 bovine serum albumin (BSA) fractionÂ V, 100Â UÂ ml-1 penicillin, 100Â ÂµgÂ ml-1 streptomycin, 2Â mM Lâglutamine, 0.1Â mM non-essential amino acids (NEAA), 0.1Â mM Î²âmercaptoethanol, MEM vitamins (1:100), 6Â mgÂ ml-1 Dâ(+)âglucose (Sigma), 30Â mgÂ ml-1 pyruvic acid, 0.1Â % DLâlactic acid (Sigma), 60Â ngÂ ml-1 progesterone (Sigma), 30Â ngÂ ml-1 Î²âestradiol (Sigma), 15Â ngÂ ml-1 mouse EGF (Peprotech), 10Â ngÂ ml-1 human bFGF (Peprotech), 10Â ngÂ ml-1 human GDNF (Peprotech), and 1000Â UÂ ml-1 mouse LIF (inâhouse preparation). For maintenance culture, GSCs were passaged at a 1:2âratio once a week after digestion with 0.05Â % trypsin/EDTA. In addition, ESCs, iPSC and gPSC were cultured on irradiated C3H feeders in the following medium: Knockout DMEM, 20% Knockout Serum Replacement, penicillin, streptomycin, Lâglutamine, NEAA, Î²âmercaptoethanol, and LIF. Generation of pluripotent cell lines. iPSC were generated using tetracycline-inducible lentiviruses. For the lentiviral production, 293T cells were plated at 50 % confluency in 10-cm dishes one day prior to transfection. The next day, the lentiviral vector (3 Âµg) together with the packaging plasmids psPAX2 (2Â Âµg) and pMD2.G (1Â Âµg) (Addgene plasmids #12260 and #12259, respectively) were transfected using FuGENE 6 reagent (Roche) according to the manufacturerâs protocol. After 48 h, viral-containing supernatants were filtered (0.45Â Âµm), concentrated around 30-fold by centrifugation (26,000 Ã g, 2Â h, 4Â Â°C) and stored at -80Â Â°C. For the generation of iPSCs, a single-cell suspension of 50,000 GSCs was infected overnight with five tetracycline-inducible lentiviruses (5â20Â Âµl concentrated supernatant each) encoding for Oct4, Sox2, Klf4, c-Myc, and the reverse tetracycline-transactivator M2-rtTA (Addgene plasmids #20323, #20326, #20322, #20324, and #20342) in the presence of 4Â ÂµgÂ ml-1 polybrene (Sigma). One week after the infection, the expression of the viral transgenes was induced by adding 4Â ÂµgÂ ml-1 doxycycline (Sigma) to the medium. MEFs-derived iPSCs were generated using the same protocol. gPSC were generated following a previous reported protocol. Briefly, GSCs were seeded at a density of approx. 1000 cells cm-2 on feeder cells and were not splitted until gPSC colonies were observed. Both iPSC and gPSC colonies were identified by Oct4-GFP expression, manually picked and expanded under ESC conditions. In vitro differentiation of pluripotent cells. Embryoid body (EB) formation was induced in suspension culture adopting the hanging drop method (600 cells in 20 Âµl) for 5 days in ESC medium without LIF. EBs were seeded on gelatine-coated plates and further cultured for 14 days in the same medium. Chimera formation. Aggregations of putative pluripotent cells with denuded 8-cell stage embryos were performed as previously reported. After overnight incubation at 37 Â°C and 5 % CO2, 11â14 aggregates were transferred into the uterine horns of 2.5 d.p.c pseudo-pregnant surrogate mothers (mated with vasectomized males). The chimeric pups were obtained and dissected at 14.5 d.p.c. Microarray gene expression analysis. Purified cRNA samples were prepared with the linear TotalPrep RNA Amplification Kit (Ambion) from 500 ng original RNA, involving synthesis of double-stranded cDNA by T7-promoter-linked oligo(dT) primers and 14 hours of in-vitro transcription incorporating biotin-labelled nucleotides. Hybridizations onto MouseRef 8 v2.0 Expression BeadChips (Illumina) was performed according to the manufacturerâs recommendations. Two replicates were hybridized from each sample. Chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using an iScan reader (Illumina). The bead intensities were mapped to gene information using BeadStudio 3.2 (Illumina). Background correction was performed using the Affymetrix robust multiarray analysis (RMA) background correction model. Variance stabilization was performed by log2 scaling, and gene expression normalization was calculated with the method implemented in the lumi package of R-Bioconductor. Samples were hybridised as biological replicates. 26 samples were analyzed. iPSC[GSCneonatal]#1: iPSC day 10 from GSC, clone 1, 2 biological rep iPSC[GSCneonatal]#2: iPSC day 10 from GSC, clone 2, 2 biological rep iPSC[GSCadult]#1: iPSC day 40 from GSC, clone 1, 2 biological rep iPSC[GSCadult]#2: iPSC day 40 from GSC, clone 2, 2 biological rep gPSC[GSCneonatal]#1: gPSC day 10 from GSC, clone 1, 2 biological rep gPSC[GSCneonatal]#2: gPSC day 10 from GSC, clone 2, 2 biological rep gPSC[GSCadult]#1: gPSC day 40 from GSC, clone 1, 2 biological rep gPSC[GSCadult]#2: gPSC day 40 from GSC, clone 2, 2 biological rep ESC: OG2 ESCs, 2 biological rep iPSC[MEF]: iPSC (Tet-4F) from OG2 MEFs, 2 biological rep GSC[neonatal]: GSC day 10, 2 biological rep GSC[adult]: GSC day 40, 2 biological rep MEF: Mouse embryonic fibroblasts, 2 biological rep

Dataset Information

Dissecting the specific role of Oct4 during the early stage of reprogramming

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets