title: NextSeq 500 sequencing; Genome wide analysis of STAT3-mediated transcription and binding sites in mouse female germ stem cell (ChIP-seq)
design: Genome wide analysis of STAT3-mediated transcription and binding sites in mouse female germ stem cell (ChIP-seq)
library_construction_protocol: FGSCs at passage number 35–45 were cultured on gelatinized plates with minimum essential medium (MEM)-α (Gibco) containing 10% fetal bovine serum (Gibco), 1mM non-essential amino acids (Gibco) , 1mM sodium pyruvate (Gibco), 2mM L-glutamine (Gibco), 0.1mM β-mercaptoethanol (Sigma-Aldrich), 10ng/ml mouse epidermal growth factor (Pepreotech), 20ng/ml mouse leukemia inhibitory factor (Pepreotech), 10ng/ml human glial cell line-derived neurotrophic factor (Pepreotech), 10ng/ml human basic fibroblast growth factor (Pepreotech), and 100units/ml Penicillin-Streptomycin Solution at 37 °C in a 5 % CO2 atmosphere as previously described (Kang Z, et al. Nat Cell Biol. 2009). cells were covalently cross-linked by incubating cells in 1 % formaldehyde (Sigma-Aldrich) for 10 minutes at room temperature and incubated with 125 mM glycine for 5 minutes to quench cross-linking reaction. The fragmented chromatin fragments were immunoprecipitated with Protein A+G Magnetic beads (Millipore) coupled with anti-STAT3 antibody (13-7000, Invitrogen). After reverse crosslinking, ChIP and input DNA library were constructed with a NEBNext Ultra II DNA Library Prep Kit for Illumina (E7645, NEB).
attributes.tag: Experimental Factor: immunoprecipitate
attributes.value: input DNA