Hybond nx

Genomic DNA (5 μg) was digested with XhoI for 5 h at 37°C. The digested DNA was separated on a 0.8% agarose gel overnight at 50 V. DNA in the gel was denatured for 1 h (0.4 M NaOH, 0.6 M NaCl) and neutralized for 1 h (1 M Trizma Base, 1.5 M NaCl, pH 7.4). DNA was transferred to a nylon membrane (Hybond NX, GE Healthcare) via capillary transfer in 10X SSC overnight and cross-linked to the membrane with UV light (auto X-link, Stratalinker). The membrane was pre hybridized for 5 h at 55°C in hybridization solution (PerfectHyb™ Plus Hybridization Buffer, Sigma). A telomere-specific probe was generated by random primed radioactive labeling with dATP [α-³²P] (DECAprime kit II; Thermo Scientific) of a double-stranded DNA fragment obtained by digestion of pBL423 (a kind gift from M.P. Longhese (pSP100)) with EcoRI followed by gel extraction. Hybridization was carried out overnight at 55°C. The membrane was washed twice in 2× SSC with 0.1% SDS for 5 min and twice in 0.5× SSC with 0.1% SDS for 20 min. All washing steps were performed at 55°C. The signal was detected via Typhoon FLA 9500 (GE Healthcare).

Total RNA was extracted from immature inflorescences (stages 1–12) as described in [50 (link)]. For small RNA blotting and detection, 10 to 12 μg were heated for 5 minutes at 95°C in 1,5 volume of standard formamide buffer and a constant volumes were loaded into a 15% Acrylamide (19:1 acrylamide:bis acrylamide), 8 M urea, 0,5X TBE gel and separated by electrophoresis. Samples were then electroblotted to Hybond-NX (GE Healthcare) and immobilized following a carbodiimide cross-linking procedure [87 (link)]. Hybridization was carried out in 15 ml of ULTRAhyb Buffer (Ambion) overnight at 50°C with a χ³²P-ATP labelled probe (T4 polynucleotide kinase, Promega, 60 minutes at 37°C). Membranes were washed twice in 3X SSC, 5% SDS and once in 1X SSC, 1% SDS. Oligoprobe sequences are found in [50 (link)]. Acquisition and quantification of the signal was achieved with a PMI-FX (BioRAD) phosphoimager and the Quantity One software.

We loaded 1 µg RNA mixed with Ambion gel loading buffer II (Thermo Fisher Scientific) and performed 16% urea PAGE. The gel was run at 120 V for 2 h in 0.5X TBE. RNA was transferred to a Hybond-NX (GE Healthcare) membrane using semidry transfer conditions at 250 mA for 45 min. RNA was then cross-linked to the membrane by adding 5 mL cross-linking solution adjusted to pH 8 (12 mL water, 122.5 mL 12.5 M 1-methylimidazole, 10 mL 1 M hydrochloric acid and 0.373 g 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) and incubating at 60 °C for 1 h in saran wrap. For each sRNA the membrane was pre-hybridised with ultra-hyb-oligo buffer (Thermo Fisher Scientific) at 37 °C for 1 h. We then incubated a mixture of 10 µL water, 4 µL 5X polynucleotide kinase forward buffer (New England Biolabs), 2 µL 10 mM DNA antisense oligonucleotide, 1 µL T4 polynucleotide kinase (New England Biolabs) and 3 µL γ-ATP at 37 °C for 1 h. The membrane was incubated in this buffer rotating at 37 °C overnight. Membrane was then washed three times in 0.2X SSC, 0.1% SDS before exposing on a phosphorimaging screen in a radioactive cassette (Fujifilm) followed by imaging on the FX Pro Plus molecular imager (Bio-Rad). The membrane was re-probed using antisense DNA oligos (Sigma Aldrich). RNA, U6 small nuclear 1 (RNU6-1) was used as the loading control.