GSM9543330: WT Biol Rep 2; Arabidopsis thaliana; OTHER
Once ap1 cal AP1-GR  and drmy1 ap1 cal AP1-GR plants began to bolt, they were induced with 10 μM Dexamethasone in 0.015% Silwet (vol/vol) three times at roughly 24 hour intervals. About 108 hours (4 ½ days) after the first induction, when flower buds had reached stages 2-3, small pieces of the floral meristem cluster were carefully removed and submerged in Tissue Tek® O.C.T. Compound (Sakura®) in a 10 x 10 x 5 mm cryomold. Air bubbles were carefully removed with a 200 µL pipette tip and 9-12 cluster pieces were arranged on the bottom of the cryomold. O.C.T. embedded tissue was then flash frozen in liquid nitrogen cooled isopentane (2-methylbutane) and stored at -70°C until cryosectioning. Fresh frozen floral meristem clusters stored at -70°C were equilibrated to -10°C in a Thermofischer HM 525 NX cryostat for about 30 minutes. Tissue blocks were cut into 10 micron thick sections and placed on each capture area according to the instructions in the 10x Visium User Guide. Each capture area had a section from a separate block of frozen tissue. We used 2 sections from each genotype to serve as biological replicates. Visium spatial RNAseq was performed according to the instructions in the user manual with the following notes and alterations. One cryosection slice was placed on each capture area of the Visium slide, each containing approximately 9-12 WT ap1 cal AP1-GR or drmy1 ap1 cal AP1-GR cauliflower-like clusters. After methanol fixation, H&E staining was replaced with toluidine blue staining as described above, then a coverslip was mounted with a solution of 85% glycerol, 10% RNAse free water, and 0.5% RNAseOUT, followed immediately by brightfield imaging as described above. Pre-permeabilization was performed as described above, and then the user manual was followed for the rest of the protocol using a 5 minute permeabilization time.