Illumina HiSeq 2000 paired end sequencing; Genome wide analysis of STAT3-mediated transcription and binding sites in mouse female germ stem cell (RNA-seq)
Genome wide analysis of STAT3-mediated transcription and binding sites in mouse female germ stem cell (RNA-seq)
FGSC at passage number 35–45 was derived from C57BL/6 wild-type neonatal mice as previously described (Kang Z, et al. Nat Cell Biol. 2009), and it was 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). Genome editing and knockout of Stat3 was performed using CRISPR/Cas9 system. Specific Stat3 gRNA Sequences (5'- GCAGGAATCGGCTATATTGC -3') were predicted by the algorithm as described previously, and the gRNA oligo were cloned in PX459 vector.  Total RNA of FGSC or ESC was extracted using TRIzol regent (Invitrogen) and resuspend in RNase free water. And 1ug total RNA was used to perform reverse transcription with Oligo-dT and random primer using SuperScript III reverse transcriptase(Invitrogen) Total RNA was extracted using Trizol (Ambion Life Technologies) according to the Ambion standard RNA isolation procedure. Poly A+ RNA was purified using the NEBNext Poly (A) mRNA Magnetic Isolation Module (E7490, NEB). Then the mRNA library was constructed with a NEBNext Ultr Directional RNA Library Prep Kit for Illumina (E7420, NEB).
Experimental Factor: genotype
wild type genotype