title: GSM7093763: Ishikawa E2 Rep1; Homo sapiens; OTHER
library_construction_protocol: Cells were permeabilized for five minutes with permeabilization buffer [10 mM Tris-HCl, pH 7.4; 300 mM sucrose; 10 mM KCl; 5 mM MgCl2; 1 mM EGTA; 0.05% Tween-20; 0.1% NP40 substitute; 0.5 mM DTT, protease inhibitor cocktail ml(Roche); and SUPERaseIn RNase Inhibitor (Ambion)]. The nuclear run-on was performed by adding permeabilized cells to run-on mixture [final composition was 5 mM Tris, pH 8.0; 25 mM MgCl2; 0.5 mM DTT; 150 mM KCl; 200 μM rATP; 200 μM rGTP; 20 μM biotin-11-rCTP (Perkins Elmer); 20 μM biotin-11-rUTP (Perkins Elmer); 1 U/μL SUPERase In RNase Inhibitor (Ambion); 0.5% Sarkosyl], then incubating at 37°C for 5 minutes. RNA was extracted with Trizol LS (Ambion), fragmented with 0.2 N NaOH for 8 minutes on ice, then neutralized with 0.5 M Tris, pH 6.8, followed by buffer exchange with a P-30 column (Bio-Rad). Biotinylated RNAs were enriched with Dynabeads M280 Streptavidin (Invitrogen), then RNA was extracted with Trizol (Ambion), 3′ adapters were ligated using T4 RNA ligase (NEB). Biotinylated RNAs were enriched for a second time, followed by 5′ cap repair with RppH (NEB) and 5′ hydroxyl repair with PNK (NEB). The 5′ adapter was ligated with T4 RNA ligase (NEB), followed by a third biotinylated RNA enrichment. Reverse transcription was performed with the RP1 primer. Samples were PCR amplified for 13 cycles, then cleaned up with Agencourt AMPure XP beads (Beckman Coulter)