Xist rna fish

Xist RNA-FISH was performed according to the manufacturer’s instructions (Biosearch Technologies). Growing cells were washed with PBS and fixed with 10% formaldehyde for 10 min at room temperature. After being washed twice with PBS, cells were immersed in 70% ethanol for at least 1 h at 4℃. Wash Buffer A (Biosearch Technologies, SMF-WA1-60) were added to cells and incubated at room temperature for 5 min. After incubation with the hybridization buffer (Biosearch Technologies, SMF-HB1-10) containing Xist RNA-FISH probe (Biosearch Technologies, SMF-2038–1) for 12–16 h at 37℃, cells were washed with Wash Buffer A and Wash Buffer B (Biosearch Technologies, SMF-WA1-20). Nuclei were stained with 0.5 mg/ml DAPI in Vectashield mounting medium. Fluorescence was detected and imaged using Leica TCS SP8 confocal microscope.

The hESCs, hBES-1 (P20 and P40), hPES-1 (P20 and P40), hPES-2 (P20 and P40) and hPES-2′ (P70) cells were used for RNA FISH analysis. XIST RNA-FISH (Biosearch Technologies, Novato, CA, USA) was carried out according to the protocol provided by Stellaris FISH Probes (Biosearch Technologies; https://www.biosearchtech.com/display.aspx?catid=224%2c318), as previously described (15 (link)). The XIST probe sequences are listed in Table I. hES cell colonies and hESCs were cultured on Millicell EZ slides (Cat. no. PEZGS0496; Millipore, Billerica, MA, USA). Stained colonies were examined under a fluorescence microscope (Leica DMIRE 2; Leica Microsystems GmbH).