Snap-freeze cell pellets on dry ice and store at -80☌ until use. pellet cells (spin at top speed for 10 sec at 4☌), then remove supernatant.ġ.5. Harvest cells with a cell scraper and transfer cell suspension to microtubes (eg 2 mL to each of three microtubes).ġ.4. Irradiate once with 2 × 400 mJ/cm 2 in a Stratalinker 2400 with 365 nm bulbs, or an equivalent instrument.ġ.3.Aspirate the medium and add 6 mL of ice-cold PBS to cells growing in a 10 cm plate (enough for three immunoprecipitations).Incubate cells with 4-thiouridine for 60 min at 500 μM or 8 h at 100 mM. Alternatively, add 10 μL of 4-thiouridine (stock concentration: 100 mM) to get a final concentration of 100 μM 4SU.
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