Hydrogel Expansion and Proteinase K Digestion

Expansion of samples was performed as described elsewhere^{55 (link)}. Briefly, monomer solution (1x PBS, 2 M NaCl, 8.625% [w/w, Sigma] sodium acrylate, 2.5% [w/w, Sigma] acrylamide, 0.15% [w/w, Sigma] N,N′-methylenebisacrylamide) was mixed and cooled to 4 °C before use. Ammonium persulfate (APS, BIORAD) initiator and tetramethylethylenediamine (TEMED, Sigma) accelerator were added to the monomer solution up to 0.2% (w/w) each. Samples on coverslips were incubated with the monomer solution plus APS/TEMED in a humidified 37 °C incubator for 1 h for gelation. Proteinase K (New England Biolabs) was diluted 1:100 to 8 units/mL in digestion buffer (50 mM Tris/HCl pH 8, 1 mM EDTA, 0.5% [v/v] Triton X-100, 1 M NaCl, Sigma) and incubated with the gels fully immersed in proteinase solution overnight at 23 °C. Digested gels were next placed in excess volumes of double deionized water for 3−4 h to expand (water changed every 30 min), until the size of the expanding sample plateaued. A small piece of the expanded sample was mounted in an ATTOFLUOR chamber (ThermoFisher Scientific) on 18 mm PLL (Sigma) coated coverslips (Marienfeld) and covered with low-melting agarose (Sigma). To determine the level of sample expansion, the average size of nuclei pre- and post-expansion was measured.