title: spec-seq of A0A0N0TFX2
design: The libraries were designed by randomizing specific positions of the experimentally confirmed GSS TFBSs . To make double-stranded DNA (dsDNA) libraries, 100 pmol synthesized single strand degenerate template oligos were mixed with 125 pmol FAM-labeled reverse complement primer. A 10-second denaturation at 90  followed by a 10-min annealing and extension at 52 in presence of Taq polymerase produced double-stranded DNAs. The DNA libraries were purified by PCR purification columns (QIAGEN), after the digestion of excess ssDNA and by incubating the mixture in presence of NEB exonuclease I for 30 min at 37. Sequences of the libraries are shown in Supplementary Figures S7 and Figures S8. The two flanking fragments for high-throughput sequencing are 5- CCAGGACTGATCGGATAGTCTCGGTTCGCATTCACCGTTCAAGAT-3 and 5- ACAGAGGTCGTATCATAAGATCGGAAGAGTGGTTCAGCAGG-3.The protein-DNA binding reactions were carried out as following: 30 pmol of FAM-labeled DNA libraries were incubated with purified protein for 30 min in a 15 l reaction system. The reaction mixtures were run in 6% non-denaturing PAGE at 120 V for 11 hour. The shifted and unshifted bands were visualized by Blue Light Gel Imager (LABGIC), cut from the gels, and the DNA separately extracted, purified and sequenced by Sangon Biotech.