Lr reaction kit

Full-length genomic DNA fragments containing ∼1 kb (MBD9) or ∼1 kb (SAC3B) of promoter sequence, together with genomic gene sequences up to the annotated/major stop codon of MBD9 and SAC3B, were amplified using PCR (SI Appendix). The PCR product was cloned into the pENTR/D vector (Invitrogen) and delivered into a modified pEG destination vector containing 3×FLAG and 9×MYC tags using the LR Reaction Kit (Invitrogen). The pEG destination vectors were transformed into Agrobacterium strain AGL0, and transformed into plants using the floral dip method (40 (link)).

The human GFP-tagged OIP5 gene cloned into the pENTR 2B vector was cloned into a pAd/CMV/V5 destination vector using the LR reaction kit (Invitrogen, Carlsbad, CA, USA). pAd/CMV/V5-OIP5 linearized with PacI was transfected into 293A cells using Lipofectamine 2000 (Invitrogen). Adenoviral stock was prepared from the supernatant and was purified using an adenovirus purification kit (Clontech, Palo Alto, CA, USA). Adenoviruses encoding β-galactosidase (LacZ) served as a control.

COP1 coding sequence minus the stop codon was PCR-amplified from Col-0 cDNA using COP1_attB1_F and COP1_attB2_R primers. The gel-eluted PCR product was used for a second round of PCR amplification with the attB1 and attB2 primers. The PCR product was cloned into pDONR221 using the BP reaction kit (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions, to generate the pDONR-COP1 construct. COP1^L105A, COP1^L170A, and COP1^MUT were generated by amplifying the pDONR-COP1 DNA (as a template) with specific primer sets (Table S1) using the QuickChange™ Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA), as described previously [52 (link)]. Then, COP1 (WT), COP1^L105A, COP1^L170A, and COP1^MUT were cloned into the pMDC85 vector [53 (link)], obtained from ABRC (CD3-744), using an LR reaction kit (Invitrogen), according to the manufacturer’s instructions, to generate pMDC85-COP1, pMDC85-COP1^L105A, pMDC85-COP1^L170A, and pMDC85-COP1^MUT constructs, respectively. All of the above constructs were transformed into Agrobacterium tumefaciens strain GV3101, which was then used to transform Arabidopsis Col-0 plants using standard protocols [54 (link)]. Transgenic lines were selected on plates containing 30 μg/mL hygromycin (Duchefa) and 250 μg/mL cefotaxime (Duchefa), and were confirmed by immunoblot analysis.