RNA-seq of hybrid mouse female NSCs for X inactivation analysis
RNA-seq of hybrid mouse female NSCs for X inactivation analysis
Neural stem cells were made from cortical dissections of E14.5 embryos dissected out in Leibovitz's L-15 Medium (Gibco). The meninges and subcortical tissue were removed before the isolated forebrains were transferred into HBSS (Gibco) containing DNase I (1 mg/mL; Sigma–Aldrich) and 0.5% trypsin-EDTA (Gibco) (53). Tissues were digested at 37 °C in 5% (vol/vol) CO2 for 20 min, followed by three washes in HBSS. The tissue was then transferred and mechanically dissociated in NeuroCult NSC Basal Medium (Mouse) (StemCell Technologies) containing NeuroCult Proliferation Supplement (Mouse) (StemCell Technologies), 0.2% heparin solution (StemCell Technologies), recombinant human EGF (20 ng/mL; StemCell Technologies), and recombinant human basic FGF (20 ng/mL; StemCell Technologies). Cells were plated onto plates coated with polyornithine (15 ng/mL; Sigma) and laminin (10 ng/mL; Sigma) at a density of 200,000 cells per square centimeter. Cells were grown at 37 °C in 5% (vol/vol) CO2 incubator and passaged every 2 d using Accutase (Sigma–Aldrich) to detach the cells from the plates. RNA (and DNA) was extracted using the Zymo Research DNA/RNA MiniPrep Plus kit, according to manufacturer's instructions The libraries were constructed using the Illumina TruSeq stranded mRNA protocol, using half reactions and starting from 500 ng total RNA.