Complete embryonic neurocult proliferation media

3 independent XX and XY primary mouse neural stem cell lines were brought to single cell suspension using ACCUTASE^TM (STEMCELL Technologies), and 1.0 × 10⁶ cells were seeded into a 10 cm non-adherent culture dish. The cells were suspended in Complete Embryonic Neurocult^TM Proliferation Media (STEMCELL Technologies) supplemented with 20 ng/ml rhEGF (STEMCELL Technologies) and with the addition of a final concentration of 20 nM Testosterone Propionate (Sigma), or the corresponding volume of DMSO vehicle (Sigma). The cells were allowed to replicate for a period of 5 days in each media type prior to being prepped histone extraction. After a 5 day growth period a subset of cells that were not used for histone extractions were brought to a single cell suspension and 5 × 10⁵ cells were seeded onto 10 cm non-adherent culture dishes. These cells were suspended in Complete Embryonic Neurocult^TM Proliferation Media (STEMCELL Technologies) supplemented with 20 ng/ml rhEGF (STEMCELL Technologies) but without the addition of TP or DMSO. After an additional 5 days of growth these cells were prepared for histone extraction.

3 independent XX and XY primary mouse neural stem cell lines were brought to single cell suspension using ACCUTASE^TM (STEMCELL Technologies), and 1.0 × 10⁶ cells were seeded into a 10 cm non-adherent culture dish. The cells were suspended in Complete Embryonic Neurocult^TM Proliferation Media (STEMCELL Technologies) supplemented with 20 ng/ml rhEGF (STEMCELL Technologies) and a single addition of TP yielding a final concentration of 20 nM Testosterone Propionate (Sigma) on day 1, or the corresponding volume of DMSO vehicle (final concentration of 1.3 × 10⁻⁴%)(Sigma). The cells were allowed to replicate for a period of 5 days in each media type prior to being prepped for RNA extraction or passaged. After a 5 day growth period a subset of cells that were not used for RNA extractions were brought to a single cell suspension and 5 × 10⁵ cells were seeded onto 10 cm non-adherent culture dishes. These cells were suspended in Complete Embryonic Neurocult^TM Proliferation Media (STEMCELL Technologies) supplemented with 20 ng/ml rhEGF (STEMCELL Technologies), without the addition of TP or DMSO. After an additional 5 days these cells were prepared for RNA sequencing.