{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"submitter":["Keisuke Kaji"],"organism":["Mus musculus"],"software":["DESeq2"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-MTAB-13169"],"description":["MEFs with doxycycline (Dox)-inducible reprogramming cassette MKOS-ires-mOrange and a Nanog-GFP reporter were transduced with lentiviral Dox-inducible Ty1-BFP (control, BFP with a Ty1 tag at the N-terminus) or Ty1-Hic2 (HIC2 with a Ty1 tag at the N-terminus) expression vector with an MOI of 3. 1 day later, reprogramming was initiated by the administration of Dox. 2, 4, 6, 10 days later, mOrange expressing cells were flow sorted for RNA extraction. Nanog-GFP+ iPS cells were flow sorted from samples cultured for 15 days in the presence of Dox, and then cultured as a bulk population in the absence of Dox for 20 days before RNA extraction. In parallel, wild-type 129 MEFs with BFP or Hic2 overexpression for 3 days, E14Tg2a ES cells with BFP overexpression were also prepared as RNA-seq samples. Libraries were prepped with the NEB Ultra II stranded mRNA Library prep kit (NEB), and the sequence was carried out with NextSeq, 75SE."],"repository":["biostudies-arrayexpress"],"sample_protocol":["Library Construction - Libraries were prepped with the NEB Ultra II stranded mRNA Library prep kit (NEB).","Sample Collection - For control and Hic2 overexpressing MEF samples, 1 × 105 Cas9 TNG MKOS MEFs were transduced with either Ty1-BFP (control, BFP tagged with a Ty1 tag at the N-term end ) or Ty1-Hic2 (HIC2 tagged with a Ty1 tag at the N-term end) lentiviruses at an MOI of 3 with 8 µg/ml polybrene (Merck-Millipore) for 4 h. After 3 days in culture in MEF media, the cells were harvested and FACS-sorted to exclude dead cells with LIVE/DEAD™ Fixable Near-IR Dead Cell Stain Kit (ThermoFisher Scientific, Dilution: 1/1500). For reprogramming samples, 0.5 × 104 Cas9 TNG MKOS MEFs were mixed with 9.5 × 104 WT MEFs (129 strain) and seeded in gelatin-coated wells of 6-well plates. Cells were transduced with either Ty1-BFP or Ty1-Hic2 lentivirus at an MOI of 3 with 8 µg/ml polybrene (Merck-Millipore) for 4h, before being recovered for 24 h in MEF media. Reprogramming was initiated by the addition of a reprogramming medium. Cells were harvested at day 2, day 4, day 6 and day 10 of reprogramming, respectively, and a maximum of 1 × 105 of mOrange+ OSKM and Nanog-GFP+ expressing cells were sorted with the FACS AriaII (BD Biosciences) per sample. Additionally, iPSCs were generated from Cas9 TNG MKOS MEFs transduced with a tet-inducible version of the Ty1-BFP and Ty1-Hic2 lentiviral particles Nanog-GFP+ iPSCs were harvested at day 15, and sorted with the FACS AriaII (BD Biosciences) and put in culture in ESC medium for 20 days in absence of doxycycline.","Nucleic Acid Extraction - All cells were homogenized with the QIAshredder kit (Qiagen) and total RNA was extracted from all samples using the RNeasy Plus Micro Kit (Qiagen).","Sequencing - RNA-sequencing was carried out with Illumina NextSeq, 75SE"],"figure_sub":["Organization","MINSEQE Score","Assays and Data","Processed Data","MAGE-TAB Files"],"data_protocol":["Data Transformation - For each sequencing run, a quality control report was generated using FastQC and Illumina TruSeq adapter sequences were removed using Cutadapt (Martin, 2011). Sequencing runs from the same biological sample were then concatenated and mapped to the GRCm38 reference genome using STAR (Dobin et al., 2013). For each biological sample, aligned sequencing reads were first assigned to genomic features (e.g., genes) using Rsubread (Liao et al., 2019) and a count table was generated. Differential expression analysis was then performed with DESeq2 (Love et al., 2014), and statistically significant genes (e.g., FDR < 0.05 and log2FoldChange > 1) were identified using the standard workflow. For exploratory analysis and visualization, a batch-corrected and regularized log matrix of expression values was used. The count table was first transformed to stabilize the variance across the mean using the rlog function from DESeq2 and then unwanted batch effects (e.g., library preparation date) were removed using the removeBatchEffect function from limma (Ritchie et al., 2015)."],"omics_type":["Metabolomics","Unknown","Transcriptomics","Genomics","Proteomics"],"instrument_platform":["NextSeq 500"],"study_type":["RNA-seq of coding RNA"],"species":["Mus musculus"],"pubmed_authors":["Keisuke Kaji"],"additional_accession":[]},"is_claimable":false,"name":"bulk RNA-sequencing of mouse embryonic fibroblast (MEF) reprogramming time course with control blue fluoresce protein (BFP) or Hic2 overexpression","description":"MEFs with doxycycline (Dox)-inducible reprogramming cassette MKOS-ires-mOrange and a Nanog-GFP reporter were transduced with lentiviral Dox-inducible Ty1-BFP (control, BFP with a Ty1 tag at the N-terminus) or Ty1-Hic2 (HIC2 with a Ty1 tag at the N-terminus) expression vector with an MOI of 3. 1 day later, reprogramming was initiated by the administration of Dox. 2, 4, 6, 10 days later, mOrange expressing cells were flow sorted for RNA extraction. Nanog-GFP+ iPS cells were flow sorted from samples cultured for 15 days in the presence of Dox, and then cultured as a bulk population in the absence of Dox for 20 days before RNA extraction. In parallel, wild-type 129 MEFs with BFP or Hic2 overexpression for 3 days, E14Tg2a ES cells with BFP overexpression were also prepared as RNA-seq samples. Libraries were prepped with the NEB Ultra II stranded mRNA Library prep kit (NEB), and the sequence was carried out with NextSeq, 75SE.","dates":{"release":"2025-04-26T00:00:00Z","modification":"2023-11-22T23:19:37.198Z","creation":"2023-07-24T09:41:03.616Z"},"accession":"E-MTAB-13169","cross_references":{"ENA":["ERP149717"],"Biostudies":["E-MTAB-13029"],"EFO":["EFO_0002944","EFO_0004170","EFO_0005518","EFO_0003816","EFO_0003738","EFO_0004184"]}}