Project description:10 million cells were used for each sample. The cells were pre-treated with or without Doxorubicin (2µM for 4h) before collection and washed with ice-cold 1 × PBS. The nuclei pellet was further fractionated into nucleoplasmic fraction (supernatant) and chromatin pellet by using MWS buffer. All the buffers above were supplemented with protease inhibitor cocktail (Thermo Fisher Scientific) and Ribonuclease Inhibitors (Promega). The RNA precipitation solution (RPS) buffer was added into the cytoplasmic fraction and nucleoplasmic fraction immediately and each fraction was stored at -20°C for at least 1h. The two fractions were centrifuged and the resulting pellets were washed with 75% ethanol. All the pellets from the chromatin fraction, nucleoplasmic fraction and cytoplasmic fraction were added with 1mL TRIzol (Invitrogen) to extract RNA. The RNA pellet in TRIzol was fully dissolved by adding EDTA to the final concentration of 5mM and heat it to 65°C. The resulting RNAs from each fraction were subjected to DNase I (Sigma-Aldrich) treatment followed by phenol/chloroform extraction to remove any contaminant DNA. The RNA pellets of each fraction were then dissolved in equal volumes of RNase-free water and used for snoRNA qRT-PCR analyses.

2021-05-04 | GSE173730 | GEO

Quantitative in vivo analysis of chromatin binding of Polycomb and Trithorax group proteins reveals retention of ASH1 on mitotic chromatin.

Project description: Not available

| S-EPMC3664806 | biostudies-other