GSM8223740: INPUT100_DAP; Medicago truncatula; OTHER
Genomic DNA was extracted from wild-type roots 14 days post-inoculation with S. meliloti strain 1021 and sonicated into ~250-bp fragments by Bioruptor Plus at 4°C. The DNA library was constructed using the NEXTflex-Rapid DNA-seq Kit (Beckman Coulter). The coding sequence of MtNIN was cloned into pFN19K HaloTag® T7 SP6 Flexi® vector with HaloTag fused in the N-terminus. The Halo-tagged MtNIN protein was expressed in an invitro TNT SP6 Wheat Germ Master Mix system (Promega) at 25°C for 2h and purified using the magnetic HaloTag beads (Promega). Subsequently, the purified protein was incubated with the prepared DNA library at 25°C for 1h, followed by washing away the unbound DNA fragments with PBS. Thirty microliters of 10 mM Tris–HCl (pH 8.5) buffer was added to the beads and incubated for 10 min at 98°C to elute the bound DNA fragments from the protein. The recovered DNA was PCR-amplified and indexed using the KAPA HiFi HotStart ReadyMix Kit (KAPA). Input DNA libraries were diluted 100 times to prepare as described above to control for background noise. The concentration of the indexed DNA was detected by qPCR and normalized. The normalized DNA library was then sequenced using the Illumina NovaSeq 6000 sequencing platform in Shanghai Hanyu Bio-Tech (Shanghai, China).