Mus musculus
Cl36 mouse embryonic stemm cells (mESCs) were cultured on 0.2% gelatin (Sigma) coated dishes at 37 °C with 5% CO2 in ESC medium: DMEM (Gibco) supplemented with 15% fetal bovine serum (FBS, Gibco), 25 mM HEPES (Gibco), 2 mM glutaMAX (Gibco), 0.1 mM non-essential amino acids (Gibco), 0.1 mM β-mercaptoethanol (Sigma), 500 units/ml leukemia inhibiting factor (LIF, Millipore), 3 μM CHIR99021 (Sigma),  1 μM PD0325901 (Sigma), and 2 μg/ml penicillin-streptomycin (Gibco). For Xist induction, 1 μg/ml doxycycline were added to the medium. For selection, 2 μg/ml puromycin (Millipore) were added.  To express cas9 in Cl36, cells were transduced with lentivirus containing EF1α-3 × FLAG-NLS-Cas9-T2A-bsd(R). 10 μg/ml blasticidin (Millipore) was added into culture medium for 3 days to select cells expressing cas9. Cas9-cl36 cells were transduced at MOI of 0.1 with lentivirus containing scramble sgRNA or individual Xist sgRNA. 4 days post-transduction, cells were divided into puromycin-containing ESC medium and were treated with either 1 μg/ml doxycycline (dox+) or DMSO (dox−). For scrambled sgRNA control, RFP+ cells were FACS sorted without dox/puro selection. For 14 Xist-derived sgRNAs, RFP+ cells were FACS sorted 7 days after dox/puromycin selection.