Nuclei of AX2 cells were isolated by sucrose gradient centrifugation as described before 60 (link) with modification in cell lysis. Here, 5 x 108 cells were resuspended in 20 ml lysis buffer (50 mM Tris pH 7, 5 mM MgCl2, 20 mM KCl, 10 % sucrose, 1 mM dithiothreitol, 0.2 mM phenylmethylsulphonyl chloride) and disrupted mechanically by the use of a cell homogenizer (Isobiotec, Heidelberg) with a clearance of 10 µm.
Nuclear run on reactions were carried out as described previously.61 (link) For inhibition of RNA Polymerase II, α-Amanitin (Applichem) was added to a concentration of 330 ng/ml for 15 minutes or 30 minutes, before addition of [32P]UTP (Hartmann). RNA was extracted with 1 ml Trizol® (Life technologies)/0.2 ml chloroform. The aqueous phase was filtered through Sephadex G50 and RNA was denatured at 95°C prior to slot blot hybridization. For the slot blot, 100 pmol of each DNA oligonucleotide probe (Sigma Aldrich) were transferred to nitrocellulose filters by using a vacuum slot blot apparatus. Oligonucleotides were then chemically crosslinked as described62 (link) and hybridized with radioactively labeled run-on RNA at 42°C over night. After three washing steps with 0,1%SDS/0,1xSSC, filters were exposed to Phosphoimager plates for 24 h.
Nuclear run on reactions were carried out as described previously.61 (link) For inhibition of RNA Polymerase II, α-Amanitin (Applichem) was added to a concentration of 330 ng/ml for 15 minutes or 30 minutes, before addition of [32P]UTP (Hartmann). RNA was extracted with 1 ml Trizol® (Life technologies)/0.2 ml chloroform. The aqueous phase was filtered through Sephadex G50 and RNA was denatured at 95°C prior to slot blot hybridization. For the slot blot, 100 pmol of each DNA oligonucleotide probe (Sigma Aldrich) were transferred to nitrocellulose filters by using a vacuum slot blot apparatus. Oligonucleotides were then chemically crosslinked as described62 (link) and hybridized with radioactively labeled run-on RNA at 42°C over night. After three washing steps with 0,1%SDS/0,1xSSC, filters were exposed to Phosphoimager plates for 24 h.
