Name: GSM5979752
Instrument: Illumina HiSeq 2000
Strategy: SELEX
Source: GENOMIC
Selection: other
Layout: SINGLE
Construction protocol: SELEX-seq dsDNA libraries were generated from ssDNA sequences by a single-cycle PCR amplification round with complementary primers essentially as described by Jolma et al. (2010). dsDNA sequences contain a 40 bp random sequence flanked by barcodes needed for later characterization after multiplex sequencing. In addition, the dsDNA sequences contained all features needed for direct sequencing on an HiSeq 2000 sequencer (Illumina). First, purified FUL antibodies resuspended in 1X PDS were coupled to magnetic beads according to manufacturer's instructions (MyOne, Invitrogen). As for EMSA, proteins were synthesized using TNT SP6 Quick Coupled Transcription/ Translation System (Promega) according to manufactures instructions in a total volume of 20 µl. Binding reaction mix was prepared essentially as for EMSA experiments with a total volume of 120 µl (Smaczniak et al., 2012b), the mix contained 20 µl in vitro synthesized protein and 50-100 ng dsDNA. The binding mix was incubated on ice for 1 hour. Followed by immunoprecipitation using 0.5 mg FUL-antibody coupled to magnetic beads (MyOne, Invitrogen) in a thermomixer at 4ºC at 700 rpm. After immunoprecipitation, magnetic beads were washed 5 times with 150 µl binding buffer without salmon-sperm DNA. Bound DNA was elucidated in 50 µl 1X TE in a 90ºC thermomixer at full speed. Next, magnetic beads were immobilized and supernatant transferred to a new tube. To allow a second round of SELEX, DNA fragments were amplified with 8 to 16 cycles of PCR with SELEX round specific primers (Jolma et al., 2010). The amplification efficiency was checked on agarose gel by comparison to a sample of known concentration. The total amplicon was used for a subsequent round of SELEX. Round for sequencing were cut out from gel after PCR amplification using MinElute Gel Extraction Kit (Qiagen). Multiple SELEX samples were multiplexed in quimolar amounts, sequencing was performed in a HiSeq 2000 sequencer (Illumina).