Nucleolar Isolation from HeLa Cells

Nucleoli were isolated after a protocol adapted from Mitrea et al. (2016) (link). 4 × 10⁷ HeLa cells were treated overnight as indicated in the figure legends and collected by trypsinization. Cells were washed in 10 ml PBS and centrifuged at 400 g for 5 min. Cell pellets were resuspended in five pellet volumes of nucleolar isolation buffer (NIB; 10 mM Tris, 2 mM MgCl₂, and 0.5 mM EDTA) with complete protease inhibitor (Roche) and incubated at RT for 2 min and then on ice for 10 min. Plasma membranes were disrupted by the addition of 10% (vol/vol) IGEPAL to a final concentration of 1% and mixed by gentle pipetting. Nuclei were isolated by centrifugation at 500 g for 3 min. The supernatant was removed and stored as the cytoplasmic fraction, and the pellets were washed in 10–15 pellet volumes of NIB and 1% IGEPAL. Nuclear pellets were then resuspended in 10 pellet volumes of NIB and sonicated on ice at 20% power for 12 cycles of 1 s on followed by 5 s off on an XL 2020 sonicator fitted with a microtip probe (Misonix). The samples were centrifuged, and the supernatant was stored as the nucleoplasmic fraction. The pellet was washed once more with 10–15 pellet volumes of NIB and finally resuspended in 50–100 µl NIB.