Transscript 2

Total RNA was extracted from 4-week-old mature leaves with Trizol reagent (Invitrogen). 50 μg of RNA enriched for small RNAs was separated by acrylamide gel electrophoresis, followed by Northern blot analysis according to published protocols^{44 (link)}. To detect small RNAs at the target regions, a PCR fragment was labeled with 32P-α-dCTP by using the Random primer DNA labeling kit (Takara). The miR167, miR159, U6 and LNA were probed with end-labeled oligonucleotides by T4 polynucleotide kinase (NEB). Northern blot signals were detected with a phosphor imager (Fuji). For RT- and qRT-PCR, total RNA was treated with Turbo DNA-free (Ambion) and reverse transcribed by TransScript II (TransGen Biotech) with gene specific primers or oligo (dT) primer. For detection of antisense transcripts, 2 pmol of forward primer was used for reverse transcription reaction in 20 μl volume. The sequences of the primers and oligonucleotides are listed (Supplementary Table S1).

For RT- and qRT-PCR, total RNA was extracted form 10-day-old or 4-week-old plants by using RNeasy Plant mini kit (Qiagen), treated with Turbo DNA-free (Ambion), and reverse transcribed by TransScript II (TransGen Biotech) with oligo (dT) primer. Then 1 μL of RT product was used as template for expression analysis. The raw data of some of the qPCR analysis are shown in Supplemental Fig. 9.