SELEX-seq dsDNA libraries were generated from ssDNA sequences which contain 20 random nucleotides in the middle by a single-cycle PCR amplification round with complementary primers essentially as described by Jolma et al. (2010). The coding sequence of AP1 was cloned into the pTNTTM vector (Promega) which was fused by 3X FLAG-tag, and then the AP1 proteins with 3XFLAG tags were synthesized using TNT® SP6 High-Yield Wheat Germ Protein Expression System (Promega) according to manufacturers' instructions in a total volume of 20 µl. The protein-DNA binding reaction mix was prepared as studied before with a total volume of 120 µl (Smaczniak et al., 2012b, Chen et al., 2023), the mix contained 20 µl in vitro synthesized protein and 50 ng dsDNA. The binding mix was incubated on ice for one hour, followed by immunoprecipitation using 10 µl Anti-FLAG® M2 magnetic beads (Sigma-Aldrich) in a thermomixer at 4ºC at 800 rpm for one hour. After immunoprecipitation, magnetic beads were washed 5 times with 150 µl binding buffer without salmon-sperm DNA and briefly rinsed with 500 µl 1XTE buffer. Bound DNA was elucidated in 50 µl 1X TE in a 90ºC thermomixer at full speed for 20 min. Next, magnetic beads were immobilized and the supernatant was transferred to a new tube. To allow a subsequent round of SELEX, the eluate DNA was amplified by PCR with a qPCR-selected cycle with SELEX PCR primers (Chen et al., 2023). The quality and concentration of the amplicon were checked by TapeStation (Agilent) and Qubit® Fluorometer (Thermo). 50 ng of the amplicon was used for the subsequent round of SELEX. For high throughput sequencing, round-specific adaptor pairs that contain all features needed for direct sequencing were used for library amplification by PCR, and the PCR cycle number used here was also selected by qPCR. All the SELEX sequencing libraries were mixed in equimolar amounts, sequencing was performed on NovaSeq 6000 (Illumina).