RNeasy Plus Mini Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
		- Include DNAse treatment for 15-20min. - Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C. - Use water to elute the RNA that is warmed to ~60`C.

Downstream tips
- Include DNAse treatment for 15-20min. - Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C. - Use water to elute the RNA that is warmed to ~60`C.

Publication protocol

U2OS cells were synchronized using a double-thymidine protocol or a thymidine–nocodazole protocol. Briefly, 3.0 × 105 cells were plated in 16 ml of DMEM. After 24 h of growth, thymidine was added to a final concentration of 2.5 mM. After 18 h in thymidine media cells were washed twice with prewarmed CO2-equilibrated phosphate-buffered saline (PBS) and allowed to grow for 8 h in prewarmed CO2 equilibrated DMEM. Again thymidine was added to a final concentration of 2.5 mM for another 18 h. Cells were washed twice with PBS and released into DMEM. For the thymidine–nocodazole synchronization, U2OS cells were plated (5.0 × 105 cells) and allowed to grow for 24 h. Thymidine (2.5 mM) was added for 18 h before cells were washed twice with prewarmed CO2-equilibrated PBS before treatment with DMEM supplemented with 100 ng/ml nocodazole for 12 h. Floating cells were collected and spun down, washed twice with prewarmed CO2-equilibrated PBS, and resuspended in prewarmed CO2-equilibrated DMEM. Nonfloating cells were washed twice with prewarmed CO2-equilibrated PBS and released into prewarmed CO2-equilibrated DMEM, and the resuspended floating cells were added back to each plate. Cells were collected every 2 h for a minimum of 36 h using RNeasy Plus Mini Kit (Qiagen, Valencia, CA). Zero-hour samples were collected while cells were still in arrest conditions.

Reference RNA was isolated from asynchronously growing U2OS cells using an RNeasy Plus Mini Kit. The same reference was used for all hybridization experiments.

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Manufacturer protocol

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