Harrington, Andrew McKain, Michael Michalski, Daniel Bauer, Kaylyn Daugherty, Joshua Steiniger, Mindy 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) Dicer2;mRNA 3’ end processing;Endogenous small interfering RNA biogenesis;Core cleavage complex;Symplekin;CPSF73 2017-04-17
    https://springernature.figshare.com/articles/journal_contribution/Additional_file_3_of___Drosophila_melanogaster_retrotransposon_and_inverted_repeat-derived_endogenous_siRNAs_are_differentially_processed_in_distinct_cellular_locations/4884101
10.6084/m9.figshare.c.3744308_D3.v1