GSM2522791: AG-AGe2-R1; synthetic construct; SELEX
The dsDNA libraries were made from the ssDNA sequences by single-cycle PCR amplification with a complementary primer essentially as described before (Jolma et al., 2010). The dsDNA libraries contained 20 random nucleotide fragment flanked by specific barcodes that allowed for later characterization when multiplexed in high-throughput sequencing. Protein dimers were synthesized using TNT SP6 Quick Coupled Transcription/Translation System (Promega) following the manufacturer’s instructions in a total volume of 20 µl and equimolar expression plasmid concentrations. The binding reaction mix was prepared essentially as described previously for EMSA experiments (Egea-Cortines et al., 1999; Smaczniak et al., 2012b) and contained 20 µl of in vitro-synthesized proteins and 50-100 ng of dsDNA library in a total volume of 120 µl. The binding reaction was incubated on ice for 1 h followed by 1 h immunoprecipitation with protein specific antibodies (affinity-purified peptide antibodies, Eurogentec) coupled to magnetic beads (MyOne, Invitrogen) in a thermomixer at 4 °C with constant mixing at 700 rpm. Magnetic beads with attached antibodies where prepared in advance according to manufacturer’s instructions (MyOne, Invitrogen) with purified antibodies resuspended in 1X PBS (~1 mg/ml); 20 µg of antibodies and 0.5 mg of beads were used for a single binding reaction. After immunopreciptiation, beads were washed 5 times with 150 µl of binding buffer without salmon-sperm DNA and bound DNA was eluted with 50 µl 1X TE in a thermomixer at 90 °C with full mixing speed. Afterwards, magnetic beads were immobilized and the supernatant was transferred to a 1.5-ml tube. DNA fragments were amplified with 10 to 15 cycles of PCR with SELEX round-specific primers (Jolma et al., 2010) and the total amplicon was used in the subsequent SELEX round. The amplification efficiency was checked on the agarose gel by comparing to a known concentration of a standard probe. Samples for sequencing, after amplification, were cut out from agarose gel and purified using MinElute Gel Extraction Kit (Qiagen). Each SELEX library was quantified by Qubit and processed by BioAnalyser. The same rounds of SELEX were multiplexed by mixing in equimolar amounts and diluted to 10 nM. Such multiplexed libraries were processed following Illumina's "Denature and Dilute Libraries Guide" with 40% PhiX control. Samples were sequences on GAII or HiSeq2000.