title: 454 sequencing of human nucleoli-associated DNA fragment library
design: Isolation of nucleolus-associated DNA (based on Sullivan et al., EMBO J (20) 2867-77):    Cells (2 x 150 mm dishes) were grown to ~70% confluence and fixed by addition of formaldehyde to the medium to a final concentration of 1%. After incubation at room temperature for 10 min, the medium was removed and the cells were washed in phosphate-buffered saline (PBS) and harvested by scraping into a total volume of 40 ml of PBS. After centrifugation at 200 g for 5 min, the cell pellet was resuspended into 1.0 ml of high magnesium buffer [HM: 10 mM HEPES pH 7.5, 0.88 M sucrose, 12 mM MgCl2, 1 mM dithiothreitol (DTT) plus protease inhibitors]. Nucleoli were released by sonication on ice (2-3 bursts of 10 s each at full power) using a Soniprep 150 (MSE) with a fine probe. Release of nucleoli was monitored microscopically. Following sonication, nucleoli were pelleted by centrifugation in a microfuge (15 000 g for 20 s). The nucleolar pellet was resuspended in 0.5 ml of low magnesium buffer (LM: 10 mM HEPES pH 7.5, 0.88M sucrose, 1 mM MgCl2, 1 mM DTT plus protease inhibitors). Nucleoli resuspended in LM were subjected to a further sonication (10 s full power) and pelleted as before. The DNA associated with the isolated nucleoli was extracted and sequenced. The isolated nucleolar DNA was prepared for 454 (GS FLX machine) sequencing by emulsion PCR and standard 454 library construction protocol at MWG-Biotech.