Lightcycler 480 green 1 master mix

For the pFtGLDP::OsCGA1 transgene expression (Figure 1b), 2 cm of mid and tip regions of emerging fourth leaves were harvested into round‐bottom microfuge tubes and frozen in liquid N₂. For dCas9‐mediated OsCGA1 activation, 2 cm sections of the mid region of the leaf were used (Figure 4g). Frozen tissues were ground by bead beating in a paint shaker with liquid N₂. RNA was extracted using TriPure isolation reagent (Roche Diagnostics, Basel, Switzerland) and 1 μg RNA of individual samples were used for residual gDNA removal using RQ1 RNase‐Free DNase (Promega, Madison, USA). After ethanol precipitation, DNase‐treated RNA was used for cDNA synthesis using Improm‐II™ reverse transcriptase (Promega) and Oligo(dT)15 primer (promega) according to the manufacturer’s instructions. cDNA was used for the RT‐qPCR using a LightCycler®️ 480 Green I Master mix (Roche Diagnostics) and primer set (Table S3) for target and reference genes on LightCycler 480 II qPCR machine (Roche Diagnostics). Os14‐4‐3E (LOC_Os11g34450) were used for normalization in Figure 1b and Os14‐3‐3E was used solely for the normalization for Figure 4g. Normalized and relative expression were calculated based on the methods in (Taylor et al., 2019 (link)).

RNA was extracted from 20 mg of frozen liver tissue preserved in RNA-later, according to the manufacturer’s guidelines (Rneasy Mini Kit, Quiagen, Venlo, the Nederlands).
cDNA was obtained from 1μg RNA using the iScript cDNA synthesis kit (Bio-Rad, Nazareth-Eke, Belgium) and real time quantitative PCR (RT-qPCR) analyses were performed using the Lightcycler 480 Green I master mix (Roche, Vilvoorde, Belgium).
Primer sets are listed in S1 Table, their efficiency was calculated from the slope of a standard curve using the following formula: $E={10}^{-\frac{1}{slope}}-1$ . All reactions were run in duplicate and normalized to reference genes that showed stable expression in all samples. The comparative Ct method was used to determine the number of transcripts.

Cells were harvested using TRIzol according to the instructions of the manufacturer. Before RNA precipitation, glycogen was added as recommended in the protocol. RNA amounts were quantified using a Nanodrop, and equal amounts were reverse transcribed following a vigorous DNase digest (Ambion, Cat # AM1906). miR‐FF4 was reverse transcribed using the Exiqon universal cDNA synthesis kit and amplified with customized primers by the Exilent SYBR Green master mix. For reverse transcription and amplification of protein‐coding genes, gene‐specific primers were designed. A list of primers can be found in the Table EV1. cDNA was generated by the Revert Aid Premium First Strand cDNA synthesis kit (Thermo) and amplified using the Light Cycler 480 Green I Master Mix (Roche).