title: GSM4852179: HIF1a ARNT round 3; synthetic construct; OTHER
library_construction_protocol: ~250nM of purified transcription factor and 200nM (1.5μl of 5μM of DNA library (Random 18mer or FixedCore 18/22mer) , ~ 7x 1010 DNA molecules) of Round 0 library were incubated at room temperature in buffer containing ~20mM Tris-HCl pH 8.0, 3mM MgCl2, ~200-300mM NaCl, 8% Glycerol, 50 μg/ml polydI-dC, 0.2 mg/ml BSA. + 10mM DTT in a 30μl for 20mins. DNA was extracted, amplified as described in Riley et al 2014. DNA isolated from SELEX Round 0 (initial Library), Round 1 and Round 2 (FixedCore 8NCGTG10N), or Round 3 (Random 18mer) were barcoded as described in Riley et al, Methods Mol Biol 2014 and illumina compatible adapters added by limited cycle PCR and cleaned up by PAGE purification. FixedCore 18/22mer SELEX samples from each round (Round 0, Round 1 , Round 2) of were quantified, pooled and run on separate lanes of a Hiseq2500 run. Random 18mer SELEX samples (Round 0 and Round 3) were barcoded by limited cycle PCR, quantified, pooled and sequenced on a NEXTSeq500 using a single end 1x75bp High Output mode. SELEX-Seq Random 18mer –   GTTCAGAGTTCTACAGTCCGACGATC(N18)CTGTCTCTTATACACATCTCCG  SELEX-Seq FixedCore 18/22mer –  SELEX_18/22mer_Multiplex1: NPAS4/ARNT2  GTTCAGAGTTCTACAGTCCGACGATCTGGNNNNNNNNCGTGNNNNNNNNNNCCAgcTgTCGTATGCCGTCTTCTGCTTG  SELEX_18/22mer_Multiplex3:SIM1/ARNT2  GTTCAGAGTTCTACAGTCCGACGATCTGGNNNNNNNNCGTGNNNNNNNNNNCCAGAACTCGTATGCCGTCTTCTGCTTG  SELEX_18/22mer_Multiplex4:HIF2a/ARNT  GTTCAGAGTTCTACAGTCCGACGATCTGGNNNNNNNNCGTGNNNNNNNNNNCCAAGAGTCGTATGCCGTCTTCTGCTTG  SELEX_18/22mer_Multiplex5:AhR/ARNT  GTTCAGAGTTCTACAGTCCGACGATCTGGNNNNNNNNCGTGNNNNNNNNNNCCAACCTTCGTATGCCGTCTTCTGCTTG