DG75-eGFP, DG75-10/12 and BCBL-1 were cultured in RPMI 1640 medium containing 100 IU/ml Penicillin, 100 µg/ml Streptomycin and 2 mM L-Glutamin. BL41, BL41 B95.8 and Jijoye were cultered in RPMI 1640 medium containing 10% fetal calf serum and Penicillin/Streptomycin as well as Bykomycine and Nystatin.
Extracted molecule
total RNA
Extraction protocol
5x10e08 cells were lyzed in 10 ml of lysis buffer containing 25 mM Tris HCl pH 7.5, 150 mM KCl, 2 mM EDTA, 0.5% NP-40, 0.5 mM DTT, and protease inhibitors (Roche); incubated for 30 min at 4°C and cleared by centrifugation at 20,000 g for 30 min. Conditions for immunoprecipitation: 60 µl Protein-G sepharose beads were incubated with 5 m RPMI containing 6µg of either anti hAgo2 (11A9) or anti-BrdU (abcam) over night. Next morning columns were drained by gravity flow and washed once with lysis buffer. Next, 5 ml of lysates were added and incubated for 3 h at 4°C. In the meanwhile, total RNA was prepared from 100µl of cell lysates. This was analyzed for BL41, B41 B95.8 and Jijoye instead of the BrdU-IP. Following the incubation, beads were washed 4x with IP wash buffer (300 mM NaCl, 50 mM Tris HCl pH 7.5, 5 mM Mg2Cl, 0.1% NP-40, 1 mM NaF) and once with PBS. RNA was recovered using miRNeasy (Qiagen).
