Additional file 3: of Drosophila melanogaster retrotransposon and inverted repeat-derived endogenous siRNAs are differentially processed in distinct cellular locations

Refined nuclear cytoplasmic/nuclear fractionation protocol. (A) S2 cells are first swelled in hypotonic buffer and then lysed with a tight-fitting dounce. Cell lysate is then centrifuged to separate the cytoplasm from the nuclei. The crude cytoplasmic fraction is purified by ultracentrifugation. The crude nuclear fraction is further purified by ultracentrifugation through a layered sucrose cushion. (B) This protocol results in excellent separation of S2 cytoplasm and nuclear material. Western blot of MEK 1/2 (cytoplasmic control) and H3 (nuclear controls) show no nuclear contamination in the cytoplasmic fraction and vice versa. (PDF 900 kb)