Pentr d topo vector

To generate the proSCAP1:GUS-GFP construct a 2977 bp region upstream of the SCAP1 start codon was amplified from genomic DNA by PCR with oligos attB1-SCAP1 and attB2-SCAP1 which contain the AttB adaptors for Gateway–mediated cloning. The PCR product was cloned into pDONR207 and subsequently transferred to pBGWFS7 destination vector [41 (link)] according to the guidelines detailed in the Gateway protocol (Life Technologies). The pro35S:amiRNA-SCAP1 constructs were engineered as detailed in http://wmd3.weigelworld.org [42 (link)] with primers I, II, III and IV. The PCR products containing SCAP1–specific amiRNA were cloned in the pENTR-DTOPO vector (Life Technologies) and transferred to the destination vector pEarleyGate 100 [43 (link)] via LR–mediated recombination. To generate the pro35S:SCAP1-YFP, the SCAP1 open reading frame (without stop codon) was amplified by PCR from Arabidopsis DNA, with primers SCAP1-Fw, SCAP1-Re2 and cloned into the pENTR-D TOPO vector (Life Technologies) and recombined with the Gateway destination vector pEarleyGate 101 [43 (link)]. The DEX-inducible SCAP1 construct (pro35S:SCAP1-GR) was kindly provided by the RIKEN Plant Functional Genomic Minami Matsui lab. Sequences of the primers are detailed in Additional file 5.

To construct the pcDNA-DEST47-miR-186 mimic, the full-length of the miR-186-5p precursor was amplified from human genomic DNA and cloned into the pENTR/D-Topo vector (ThermoFisher Scientific) with BamHI and NotI restriction enzymes. The primer sequences for miR-186-5p were: miR-186-5p forward (5’-GCggatccGAGCCATGCTTATGCTACTG-3′) and miR-186-5p reverse (5′ -GCgcggccgcCCAGGTATATGGCA-3′).
To construct the pcDNA-DEST47-anti-miR-186, the full-length of anti-sense miR-186-5p amplified from human genomic DNA of RWPE1 cells was cloned into the pENTR/D-Topo vector (Thermo Fisher Scientific) and shuttled into pcDNA-DEST47 mammalian expression vector with BamHI and NotI restriction enzymes. The anti-sense oligonucleotide sequences were: anti-miR-186-5p forward (5’ CACCGCggatccTGCTTGTAACTTTCCAAAGAATTCTCTCCTTTTGGGCTTTCTGGTTTTATTTTAAGCCCAAAGGTGAATTTTTTGGGAAGTTTGAGCT-3′) and anti-miR-186-5p reverse (5’ gcggccGCAGCTCAAACTTCCCAAAAAATTCACCTTTGGGCTTAAAATAAAACCAGAAAGCCCAAAAGGAGAGAATTCTTTGGAAAGTTACAAGCA-3′). Clones were verified via DNA sequencing by Eurofins Genomics (Louisville, KY).

The mouse Phf8 transcript (NM_177201) was purchased from Origene (Cat#: MR223276) and subcloned into a pENTR/D-TOPO vector (Thermo Fisher Scientific, Waltham, MA, USA). The mouse pseudogene Phf8 (Phf8-ps) (Ref Sequence:4921501E09Rik, NM_001009544) was cloned by PCR into a pENTR/D-TOPO vector (Thermo Fisher Scientific, Waltham, MA, USA), using mouse genomic DNA. Both Phf8 and Phf8-ps sequences were then transferred by Gateway reaction into a pHAGE-HA/FLAG vector, using the Gateway LR Clonase II enzyme (Thermo Fisher Scientific, Waltham, MA, USA).