Molecular Techniques for Arabidopsis Transcriptomics

Total RNA was isolated using the Quick-RNA MiniPrep kit (Zymo Research). Briefly, Arabidopsis seedlings (~60 mg) were ground in extraction buffer and processed following the manufacturer’s protocol. Reverse transcription was performed using M-MLV RTase (Promega) using anchored oligo dT. For qPCR, the FastStart Essential DNA Green Master mix was used with a LightCycler 96 (Roche Applied Science). Relative transcript levels were determined by normalizing with PP2A (At1g13320). Immunoblot analyses were performed using rabbit anti-RGA antiserum (DU176, 1:10,000 dilution)^{11 (link)}, horseradish peroxidase (HRP)-conjugated anti-FLAG M2 mouse monoclonal (Sigma Aldrich A8592, 1:10,000 dilution) and mouse HRP-anti-MYC monoclonal antibodies (BioLegend #626803, 1:1,000 dilution), mouse anti-HA monoclonal antibody (BioLegend 901503, 1:1,000 dilution), rabbit anti-H2A monoclonal antibody (Abcam #ab177308, 1:1,000 dilution) and rabbit anti-Histone 3 polyclonal antibody (Abcam #ab1791). HRP-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch #715-035-150) was used for anti-HA at 1:10,000 dilution. HRP-conjugated goat anti-rabbit IgG (Thermo-Fisher #31462) was used to detect anti-RGA and anti-HA at 1:10,000 dilution. Y2H assays were performed as described previously^{45 (link)}.