Stellaris rna fish wash buffer a

RNA FISH for Xist and another X-linked gene, Huwe1 was performed using Stellaris FISH probes (Biosearch Technologies). Probe details can be found in Supplementary Data 4. Cells were dissociated using Accutase (Invitrogen) and adsorbed onto coverslips (#1.5, 1 mm) coated with Poly-l-Lysine (Sigma) for 5 min. Cells were fixed with 3% paraformaldehyde in PBS for 10 min at room temperature (18–24 °C) and permeabilized for 5 min on ice in PBS containing 0.5% Triton X-100 and 2 mM Ribonucleoside Vanadyl complex (New England Biolabs). Coverslips were preserved in 70% EtOH at −20 °C. Prior to FISH, coverslips were incubated for 5 min in Stellaris RNA FISH Wash Buffer A (Biosearch Technologies), followed by hybridization overnight at 37 °C with 250 nM of each FISH probe in 50 μl Stellaris RNA FISH Hybridization Buffer (Biosearch Technologies) containing 10% formamide. Coverslips were washed twice for 30 min at 37 °C with Stellaris RNA FISH Wash Buffer A (Biosearch Technologies), with 0.2 mg/ml Dapi being added to the second wash. Prior to mounting with Vectashield mounting medium coverslips were washed with 2× SSC at room temperature for 5 min. Images were acquired using a widefield Z1 Observer microscope (Zeiss) using a 100× objective. The intronic signal of Huwe1 was used in combination with Xist to estimate the percentage of XO cells in the population, which was maximally 5%.