Maxiscript t3 kit

Embryos were collected 0 to 3hr 30 min after the egg laying. Fixation of embryos and in situ hybridization with whole mount embryos was as described (Lu et al. 2008 (link)). Embryos are mounted in 70% glycerol in PBS for imaging. Stages of embryo were detected based on number of nuclei, shape of the nuclei, and cellular furrows as outlined (Lu et al. 2008 (link)). Templates for in vitro RNA transcription was made by PCR amplification with a forward primer and a reverse primer along with T3 promoter using genomic DNA from w¹¹¹⁸ flies. A Digoxygenin labeled antisense RNA probe was synthesized using in vitro transcription kit (MAXISCRIPT T3 kit, Ambion). Probe was detected using anti-Digoxygenin antibody (Roche) that cross react with NBT-BCIP solution staining the embryos. Primers used to in vitro templates were: Sxl forward 5′-CCCTACGTCGACGGCATTGCAGC-3′, Sxl reverse 5′-TAATACGACTCACTATAGG-GAATGACCCAATGGAATCG-3′ and runt forward 5′-AACGACGAAAACTACTGCGGCG-3′, runt reverse 5′-AATTAACCCTCACTAAAACGGTCACCTTGATGGCTTTGC-3′.

For RNA-EMSAs, target sequences were amplified from potato leaf cDNA or genomic DNA using corresponding primers (Supplementary Table S4 at JXB online). These probes were derived from either coding sequence or the 3′-UTR, and were selected based on the yeast three-hybrid results (Supplementary Fig. S4). RNA bait was generated using in vitro transcription with the MAXIscript^® T3 kit (Ambion) and biotin-labeled UTP (Bio-11-UTP, Ambion). Biotin-labelled RNA probes were purified using RNA Clean & Concentrator™-5 (Zymo Research) and quantified by NanoDrop 1000 (Thermo Scientific). Biotin-labelled RNAs (3fmol) were used for the binding assay. Binding reactions with labelled RNA and purified recombinant proteins (0–500nM) were incubated in 20 µl for 1.0h at room temperature in the presence of a binding buffer consisting of 40mM Tris (pH 8.0), 30mM KCl, 1mM MgCl₂, 0.1% NP-40, and 1mM DTT. The binding reactions were resolved on a 6% (v/v) non-denaturing polyacrylamide gel, and then transferred onto a BrightStar^®-Plus positively charged nylon membrane (Ambion). For detection of biotinylated RNA, the Chemiluminescent nucleic acid detection module kit (Thermo Scientific) and CL-XPosure™ film (Thermo Scientific) were used.