Amplicon of gDNA of synthetic transcription factor library
Amplicon sequencing libraries were prepared from genomic DNA in 3 steps. First, the general vicinity of the tile sequence was amplified with CP21.P14 and CP17.P12 using 100 ng of gDNA as template and yielding a 604 bp product that was cleaned up (Monarch PCR cleanup). In the second PCR, we added 1-4 bp of phasing on each end and the Illumina sequencing primer in 7-10 cycles with SL5.F[1-4] and SL5.R[1-3]. These 7 phased primers were pooled and added to all samples. 4 ng of the first PCR was used as template for the second PCR. 2 ul of the second PCR served as template for the third PCR. The third PCR added unique Index1 and Index2 sequences to each sample with an additional 7-10 cycles. These final products were cleaned up with PCR columns or magnetic beads (MacroLab at UC Berkeley) and submitted for sequencing.