RNA Extraction and Northern Blot Analysis

RNA was extracted from leaf material harvested in low light and high light (same material as used for ribosome profiling, RNA-seq, and RNA secondary structure probing with NAI-N₃) by adding 666 µL of extraction buffer (Section 2.4) to frozen, ground material. The RNA was purified from the extract using a phenol/chloroform/isoamyl alcohol step and isopropanol precipitation. The gel blot was done as described before [51 (link)]: 10 µg total RNA was separated on 15% polyacrylamide TBE urea gels (Biorad, Hercules, CA, USA) and transferred in a wet-blot setup with 0.5 TBE buffer to a Hybond-N membrane (GE-Healthcare Life Sciences). The RNA was cross-linked to the membrane with 0.16 M N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride in 0.13 M 1-methylimidazole (pH 8.0) at 60 °C for 1 h. The probe (see Supplemental Table S4) was labelled at the 5′ end with γ-³²P-ATP and hybridized at 60 °C using standard protocols.