spec-seq of A0A0L8NT36
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 7 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.