Tbe urea polyacrylamide gels

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.

Intracellular polyP granules were examined by Neisser staining (31 ) and visualized by light microscopy. Cells grown in PNMC were fixed with paraformaldehyde (57 (link)), mounted on a slide, and stained with solution A (methylene blue, 0.1%; glacial acetic acid, 5%; ethanol, 5%) and solution B (crystal violet, 10%) for 15 s. Slides were stringently rinsed with water and allowed to dry, followed by staining with solution C (chrysoidin Y, 1%) for 45 s and stringent washing. Images were captured using a Zeiss Axioscope (differential inference contrast [DIC] setting) with a 63× oil immersion objective.
For urea-PAGE analysis, RNA and polyP were extracted as previously described (5 (link)). Twenty micrograms of RNA containing polyP was resolved on 12% polyacrylamide TBE-urea gels (Bio-Rad) in 1× Tris-borate-EDTA (TBE) buffer. Following electrophoresis, gels were fixed with glycerol and methanol, stained with toluidine blue, and destained as described previously (5 (link)).