title: GSM7889867: 10nM FOXA1, Shifted Band, Rep 1; synthetic construct; OTHER
library_construction_protocol: Synthetic oligonucleotide library was designed to include 3,203 different sequences, each 229bp in length with 193bp variable regions and 18bp primer-binding regions on the two sides. The synthetic oligo library was ordered from Agilent (Product #G7220A). To amplify the synthetic oligonucleotide library sequences, we performed two rounds of PCR amplification with a BbsI digestion step in between. The primer pairs for the first round of PCR contain regions annealing to the CCND1e (outside the 193bp library sequence) at the 3'-ends, partial Illumina TruSeq adaptor sequences at the 5'-end and 0-3 random nucleotide spacers in between to increase sequence complexity. Primers with different numbers of spacers are mixed in equimolar ratio for the first round of PCR. Preliminary PCR tests were performed to decide the optimal cycle number that keeps the PCR reactions in exponential phase. We used 23-25 cycles for our first round of PCR. For the first round of PCR, 30 μl of ChIP DNA was amplified in a 100 μl PCR reaction using NEBNext Ultra II Q5 master mix. The PCR products were purified with AMPure XP beads with a beads to DNA ratio of 0.9. 15 μL of the purified PCR product was digested with 20 U of BbsI in a 30 μL reaction at 37°C for two hours. The digestion products were purified with AMPure XP beads with a beads to DNA ratio of 0.9, followed by the second round of PCR. The primer pairs for the second round of PCR contain the rest of the Illumina TruSeq adaptor sequences and sample indexes. For the second round of PCR, 2 μl of purified digestion product was amplified in a 50 μl PCR reaction for 8 cycles using NEBNext Ultra II Q5 master mix. The PCR products were purified with AMPure XP beads with a beads to DNA ratio of 0.8. Quality control was conducted with TapeStation (Agilent). 30 million paired-end 150bp reads were obtained for each sample using a NextSeq 2000 (Illumina) instrument.