Nucleus homogenate preparation

Unless otherwise noted, all procedures should be performed at 4°C or on ice. Once prepared. Drosophila nuclei should be handled identically, regardless of source. The nucleus-enriched pellet should be resuspended in 20 mM Tris-HCl, pH 7.5,5 mM MgCla (Buffer B). For nuclear resuspension, one volume of Buffer B relative to the initial volume of starting material should be used. For example, if nuclei are purified from 10 ml of embryos, 10 ml of Buffer B should be used if nuclei are purified from 1 ml of tissue culture cells, 1 ml of Buffer B should be used. Complete resuspension should be assured by Dounce homogenization (two-four strokes, loose pestle) and both DNase I and RNase A should be added to final concentrations of 10 and 8 /i.g/ml, respectively. Typically, RNase A is added first, the sample is mixed on a vortex mixer, and then the DNase I is added. After addition of DNase I, vortex mixing is avoided because DNase I is very sensitive to inactivation by oxidation. Rather, the sample is mixed by gentle agitation. 

Preparation of oocyte samples and immunoprecipitation. Extracts containing P-labeled RNAs are prepared either from whole oocytes or isolated nuclear and cytoplasmic fractions by homogenization on ice in a suitable buffer (e.g., 40 mM HEPES, pH 7.9, with KOH 100 mM potassium acetate, 8% (v/v) glycerol, 5 mM MgS04, 2 mM EGTA, 0.2 mM EDTA, 1 mM dithiothreitol, 0.5% (v/v) aprotinin, 2 /ig/ml of each leupeptin and pepstatin, and 0.1 U/jul of RNasin) at a ratio of at least 25 pi of buffer per oocyte (or isolated cytoplasm) or as little as 1-4 pi per isolated nucleus. If the antibody recognizes RNA rather than an... 

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