biostudies-arrayexpressMetabolomicsUnknownTranscriptomicsGenomicsProteomicsMichael ZillerChIP-seqMus musculusMus musculushttps://www.ebi.ac.uk/biostudies/studies/E-GEOD-53768Pluripotent stem cells retain the ability differentiate into the three germ layers and germ line. As a result, there is a major interest in characterizing regulators that establish and maintain pluripotency. The network of transcription factors continues to expand in complexity, and one factor, Undifferentiated embryonic cell Transcription Factor 1 (Utf1), has recently moved more into the limelight. To facilitate the study of Utf1, we report the generation and characterization of two reporter lines that enable efficient tracking, mapping and purification of endogenous Utf1. In particular, we include a built-in biotinylation system into our targeted locus that allows efficient and reliable pulldown. We also use this reporter to show the dynamic regulation of Utf1 in distinct stem cell condibiostudies-arrayexpressGrowth Protocol - Mouse embryonic stem (ES) cells were cultured in DMEM media containing 15% FBS, 1% PEN/STREP, 1% Glutamine, 1% NEAA and 5x105U LIF. For ES cell maintenance dishes were coated with 0.2% gelantin and irradiated CF1 mouse embryonic fibroblasts (MEFs) were plated as confluent layer of feeder cells. ES cells were seeded in a density of 50000 cells/ 6-well and were passaged every 2-3 days. The 2i media was prepared according to the following protocol (Ying et al., 2008) and was supplemented with 5x105U LIF.Library Construction - Cells were crosslinked in 1% formaldehyde for 5 minutes at room temperature, with constant agitation, followed by quenching with 125mM Glycine for 5 minutes at room temperature. Nuclei were isolated and chromatin was sheared using Branson sonifier until the majority of DNA was in the range of 200-700 base pairs. Chromatin was incubated with antibody overnight at 4M-0C, with constant agitation. Immunoprecipitation of antibody-protein complexes was completed using Protein A or Protein G Dynabeads (Invitrogen) for 2-3 hour at 4M-0C, with constant agitation. ChIPs were completed using previously reported methods (Mikkelsen et al., 2010). For the bioChIP Streptavidin-coupled Dynabeads (Invitrogen) were incubated with the chromatin for 2-3 hours at 4M-0C. The SDS concentraSample Treatment - ChIP was performed using the following antibodies at a final concentration of 1M-5g/1x106 cells: Utf1 (Abcam, ab24273).OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesMichael ZillerAlex MeissnerData Transformation - Base calling was performed with the standard Illumina pipeline 1.83 ChIP-Seq data was aligned to the mm9/NCBI37 reference genome using bwa version 0.5.7 (Li and Durbin, 2009) with default parameter settings. Subsequently, reads were filtered for duplicates and extended by 200bp. Transcription factor ChIP-Seq data for Utf1 was first analyzed with macs version 1.4 (Zhang et al., 2008) to determine regions of enrichment using the following parameters: -g 1.87e9 --tsize=36 --pvalue=1e-5 --tsize=36 --pvalue=1e-5 --keep-dup=1 and using the KH2_mES_WCE as input control. Data analysis was conducted in R version 2.15.2 (http://www.r-project.org/) and python version 2.6.5 (http://python.org/) using custom scripts. Genome_build: mm9 Supplementary_files_format_and_content: ProcesfalseIn Vivo and In Vitro Dynamics of Undifferentiated Embryonic Cell Transcription Factor 1Pluripotent stem cells retain the ability differentiate into the three germ layers and germ line. As a result, there is a major interest in characterizing regulators that establish and maintain pluripotency. The network of transcription factors continues to expand in complexity, and one factor, Undifferentiated embryonic cell Transcription Factor 1 (Utf1), has recently moved more into the limelight. To facilitate the study of Utf1, we report the generation and characterization of two reporter lines that enable efficient tracking, mapping and purification of endogenous Utf1. In particular, we include a built-in biotinylation system into our targeted locus that allows efficient and reliable pulldown. We also use this reporter to show the dynamic regulation of Utf1 in distinct stem cell condi2014-01-03T00:00:00Z2023-09-15T01:45:00.952Z2022-03-04T17:55:21.064ZE-GEOD-53768SRP034872EFO_0002692