SELEX-Seq for ESRP motif analysis
Random 20mer RNA sequences were generated from a 103 base DNA oligonucleotide containing a T7 promoter, restriction sites, 20 random bases and an SP6 promoter sequence. The full sequence is (with N standing for randomized bases): AATTTATAATACGACTCACTATAGGGAGAAAGTTGGCCGCAGTATCGATANNNNNNNNNNNNNNNNNNNNCTCGAGTTCTATAGTGTCACCTAAATCAAGCTT  This oligo was made into a double stranded DNA library using Klenow Exo Minus (Promega). This randomized double stranded DNA pool was blunt cloned into the pCR-Blunt vector and used as a template for T7 transcription per manufacturer's instructions (Ambion). Production and purification of recombinant Glutathione-s-transferase (GST)-Esrp1 fusion protein was performed as previously described. GST-Esrp1 protein was bound to GST beads rotating at 4C for 30 minutes and RNA was also pre-cleared against GST beads. The randomized RNA pool was incubated with GST-Esrp1 at a 30-40:1 molar ratio of RNA to protein in a 200 ml binding reaction (16 mM Hepes pH 7.9, 95 mM KCl, 2 mM MgCl2, 60 uM EDTA, 1mM DTT, 6% glycerol, 0.1 mM heparin (Sigma #H-3393), and 0.15 mM PMSF). Binding reactions were incubated rotating at room temperature for 30 minutes and washed 5 times with the same binding buffer. Bound RNA was isolated from beads using Trizol reagent according to manufacturer's instructions (Invitrogen) and used for reverse transcription with an SP6 specific primer to create a cDNA library from the bound RNA. This cDNA was then used as a template for a T7 transcription. This cycle was repeated for a total of 7 rounds. In the 7th round, KCl concentration for binding and washes was increased to 300mM.    Sequencing libraries were prepared from round 0, 2, 3, 6 and 7 cDNA pools using a PCR strategy with modified Illumina adaptor sequences containing 4 nucleotide barcodes specific to each round and sequenced using the Illumina GenomeAnalyzer II to generate 44 bp single end reads.