V5 IPs for RNA enrichment

The protocol is adapted from this one, here. Briefly,

• Quench exponentially proliferating cells with 100 μg/mL cycloheximide.

• Collect cells by centrifugation and wash with freezing buffer (15% glycerol, 150 mM potassium acetate, 2 mM magnesium acetate, 20 mM HEPES/sodium hydroxide pH 7.2, 0.5% (w/v) glucose, 100 μg/mL cycloheximide).

• Resuspend in the same freezing buffer (1E+08 cells in 60 μL buffer), and store at -80 °C until ready.

• Thaw cells on ice.

• Wash cells with 1 mL of RIP buffer (150 mM KCl, 25 mM Tris pH 7.4, 5 mM EDTA, 0.5 mM DTT, 0.5% IGEPAL® CA-630, 100 μg/mL cycloheximide).

• Resuspend cells in 0.6 mL of RIP buffer containing 100 U/mL RNAse inhibitor SUPERase•in™ (ThermoFisher; Cat#: AM2694) and protease inhibitor cocktail (Millipore Sigma; Cat#: 11836170001). Add the RNAse and protease inhibitors fresh each time.

• Add about 0.250 mL of glass beads (one 'scoop') and vortex at the maximum speed for 30 sec, then place on ice for 30 sec. 

• Repeat the vortex-ice cycle for a total of six times, to break the cells.

• Collect the supernatant after a centrifugation at 5,000 rpm for 5 min.

•  Clarify the supernatant with another centrifugation at 12,000 rpm for 2 min. 

• Isolate the clarified supernatant and remove 0.15 mL ( store at -80 °C) , to serve as ‘input’ control.

•  To the rest of the supernatant, add 10 μL of agarose-α-V5 beads.

•  Incubate at 4 °C on a tube rotator for 1-2 h. 

• Pellet the beads at 1,000 rpm for 1 min.

• Slowly and carefully remove the supernatant. Don't be too greedy, leave 20-50 μL of supernatant in the tube.

• Wash the beads  with 0.5 mL RIP buffer, and pellet as before. 

• Repeat twice more the wash step.

• Resuspend the beads in 130 μL of RIP buffer and store at -80 °C, prior to RNA isolation and ddPCR.