title: P-bodies purified from PC3-LN4 cells treated with AKT inhibitor for 6 hours - Rep 3
design: Total RNA was isolated from cells and P-body samples using TRIzol reagent (Invitrogen, 15596018) followed by column extraction using Quick-DNA/RNA Microprep Plus Kit (Zymo Research) per the manufacturers protocol. Briefly, cold TRIzol was added directly to the culture dish or GFP-Trap beads following P-body purification. The samples were stored at 20C overnight, thawed on ice, and chloroform was added to the mixture and shaken. This mixture was incubated for 23 min and centrifuged at 4C for 15 min. Following separation of the mixture into the lower red phenol-chloroform, interphase, and a colorless upper aqueous phase. The upper aqueous phase was collected and added directly to the Quick-DNA/RNA Microprep Plus Kit column, and the standard protocol was followed. Paired-end Illumina RNA-sequencing was performed in triplicate