Combined XIST RNA FISH or ATRX intron RNA FISH with chromatin-tracing primary probe hybridization

The cells grown on a silanized coverslip (see the “Silanization of coverslips” section) were fixed and permeabilized as previously described in the “Chromatin tracing primary probe hybridization” section. The cells were incubated with prehybridization buffer composed of 50% (v/v) formamide in 2× SSC for 5 min at room temperature and then hybridized with 1 to 2 μM XIST RNA FISH probes or ATRX intron RNA FISH probes in hybridization buffer composed of 50% (v/v) formamide, 10% (w/v) dextran sulfate, yeast tRNA (1 mg/ml), and 1% (v/v) murine RNase inhibitor in 2× SSC and incubated in a humid chamber/petri dish at 37°C overnight. The cells were washed with 0.1% (v/v) Tween 20 in 2× SSC at 60°C water bath twice for 15 min each and once at room temperature for 15 min.

After the XIST RNA FISH or ATRX intron RNA FISH, the coverslip was embedded with 4% (w/v) polyacrylamide gel to immobilize the probes in place, preventing the detachment of the oligo probes during RNase A digestion in the chromatin tracing primary probe hybridization procedure. To embed the sample, we freshly prepared a degassed gel solution containing 4% (w/v) acrylamide and bis-acrylamide solution, 300 mM NaCl, 60 mM tris-HCl (pH 8.0), ammonium persulfate (0.3 mg/ml), 0.15% (v/v) N,N,N',N'-Tetramethyl ethylenediamine (TEMED), and 1:100,000 yellow-green fiducial beads. We prepared a 2″ by 3″ glass slide covered with gel slick solution and dried for 10 min in air. We removed the excess liquid on the glass slide before adding gel solution. We dropped 50 μl of gel solution onto the slick glass slide and then gently flipped the coverslip onto the gel solution without causing bubbles. After 1.5 hours, the polyacrylamide gel solidified, and the coverslip was gently removed from the glass slide with a razor. The sample was proceeded to chromatin tracing primary probe hybridization starting from the HCl treatment.