title: GSM1183034: EMSA-Seq of p50p50 round 3 of SELEX; Homo sapiens; ChIP-Seq
library_construction_protocol: For each round of SELEX selection, two protein binding reactions (all 10 μl) were performed simultaneously. Reaction 1 contained 200 ng 64-bp positive dsDNA, 115 ng (Reaction 2) p50 protein (Promega) in 10 mM Tris-HCl, pH7.5, 50 mM NaCl, 0.5 mM EDTA, 3 mM MgCl2, 1 % glycerol, 0.5 mg/ml BSA, 0.05% NP-40, 0.01 mM DTT. Reaction 2 contained 200 ng 64-bp random dsDNA, 115 ng p50 protein in 10 mM Tris-HCl, pH7.5, 50 mM NaCl, 0.5 mM EDTA, 3 mM MgCl2, 1 % glycerol, 0.5 mg/ml BSA, 0.05% NP-40, 0.01 mM DTT and 0.1 μg polydI-dC (Amersham). The protein binding reactions were incubated at room temperature for 60 min and loaded on 6% native polyacrylamide gel and migrated in 0.5 × TBE buffer for 1.5 hs. After electrophoresis, the gel was directly visualized on UV transilluminator and the gel slice containing the shifted dsDNA (Reaction 2) was excised according to the position of the shifted band of Reaction 1. The gel slice was socked in 100 μl diffusion buffer [0.5 M NH4Ac, 10 mM Mg(Ac)2, 1 mM EDTA, pH8.0, 0.1% SDS] overnight at 37°C. The diffusion buffer was purified with Microcon YM-30 and DNA was eluted in 25 μl ddH2O. For experiments in which several rounds of SELEX were carried out the following procedure was adopted. DNA obtained after the first round of TF-DNA binding followed by EMSA, elution and purification was amplified using a high-fidelity PCR procedure. Briefly, multiple 10 μl reactions each comprised of 1 mM dNTPs, 4μM of Primers 1 and 2 (using nested primers: R-F1 and R-R1 for Round 1, R-F2 and R-R2 for Round 2; R-F3 and R-R3 for Round 3; R-F4 and R-R4 for Round 4), together with 1 units of PrimerStar® HS Taq polymerase (TaKaRa) buffered in accompanying 1X PCR Buffer were incubated for 3min at 94°C, then subjected to 20 PCR cycles (1min 94°C, 1min 60°C, 2 min 70°C). DNA was pooled, purified with Microcon YM-30 (Millipore). This was subsequently used as starting material for TF-DNA binding in the second round of SELEX. The dsDNAs selected in Round 0 to 4 were pooled in equal molar to form a multiplexed sample for sequencing.