Fla 7000 phosphoimager

This analysis was carried out according to the protocol described in [34 (link)]. Briefly, genomic DNA was isolated from leaves of 8-week-old plants by the protocol published in [35 (link)]; approximately 2 g (fresh weight) of leaves were collected from each plant. Five µg of genomic DNA were digested by the MseI (NEB, Ipswich, MA, USA; recognition sequence T/TAA) and analyzed by Southern hybridization with a radioactively labeled telomeric probe synthesized by non-template PCR, using the modified protocol described in [36 (link),37 (link)]. Hybridization signals corresponding to telomeric tracts (plus subtelomeres up to the first restriction site upstream of telomeres) were visualized using FLA7000 phosphoimager (FujiFilm, Minato, Japan) and analyzed by the MultiGauge V2.0 software (FujiFilm). The median of telomere length was calculated as ∑(OD_i × L_i)/∑(OD_i); OD_i is the signal intensity above background within the interval i, L_i is the molecular weight (kb) at the mid-point of the interval i.

Northern blot experiments were essentially performed as described previously26 (link)56 (link). RNA was prepared with the hot phenol method and 10 μg per lane resolved on 1.2% agarose gels containing 6.7% formaldehyde in MOPS buffer. After capillary transfer in 10 × SSC onto Hybond N+ membranes (GE Healthcare), RNA was UV-crosslinked and stained with methylene blue. Gene-specific probes were generated by random priming in the presence of ATP[α-³²P] using the Prime-It II Random Primer Labeling Kit (Agilent) and hybridized at 42 °C overnight. After repeated washes in 2 × SSC, 0.1 % SDS, blots were exposed on Amersham Hyperfilm MP (GE Healthcare) or quantified with a Fla-7000 phosphoimager (Fujifilm). For adh1, strand-specific, digoxigenin (DIG)-labelled probes were used which were generated by in vitro transcription with the MAXIscript kit (Ambion) and detected using the DIG system (Roche). Uncropped versions of the northern blots are shown in Supplementary Figs 11 and 12.

Analysis was performed according to the protocol described in [7 (link)] with modifications presented in [20 (link)]. Genomic DNA from seedlings cultivated on three Petri dishes and from leaves of three plants grown from these seedling were isolated, as described in Section 4.2 and Section 4.4, respectively. For bisulfite conversion (described in Section 4.4), DNAs from respective organs were mixed in equimolar ratio. Alternatively, DNA isolated from seedlings taken from one Petri dish or leaves collected from one plant was treated by bisulfite. Then, 500 ng of DNA was transferred to the Hybond XL membrane (GE Healthcare, Chicago, IL, USA) and hybridized against radioactively labeled probes in ULTRAhybTM-Oligo Hybridization Buffer (Thermo Fisher Scientific, Waltham, MA, USA). The probe (TTAGRRT)₄, R = A or G, was designed to hybridize with the fraction of telomeric repeats in which the inner cytosine was methylated and the other cytosines were either methylated or non-methylated; (ACCCTAA)₄ probe hybridizing with the telomeric G-strand was used for DNA loading normalization. Hybridization signals were visualized using the FLA 7000 phosphoimager (FujiFilm) and evaluated using the MultiGauge software (FujiFilm).

SB100X or the I212S mutant were mixed with LO52 (20 nM) at a molar ratio of 10:1 in 20 mM Hepes (pH 7.2), 150 mM NaCl, 10 mM MgCl₂ and 1 mM DTT. Reactions were incubated at 25 °C for 20 h and terminated by Proteinase K (New England Biolabs) treatment according to the manufacturer's instructions. Reaction products were ethanol-precipitated, re-suspended in 2 × formamide loading dye and analysed by gel electrophoresis on TBE-urea 12% acrylamide/bis-acrylamide (19:1) gels. Gels were imaged on a FLA 7000 phosphoimager (Fuji) and quantified in the Fujifilm Multi Gauge software package.

Total RNA was extracted by a hot phenol method. 10 μg of RNA was loaded per lane and resolved on 1.2% agarose gel containing 6.7% formaldehyde in MOPS buffer. After capillary transfer in 10 × SSC onto Hybond N+ membranes (GE Healthcare), RNA was UV-crosslinked and stained with methylene blue. Gene-specific probes were generated by PCR using oligos listed in Table S7 and labeled by random priming in the presence of [α-³²P] ATP using the Prime-It II Random Primer Labeling Kit (Agilent). The membrane was hybridized at 42 °C overnight. After repeated washes in 2 × SSC, 0.1% SDS, blots were exposed on Amersham Hyperfilm MP (GE Healthcare) or quantified with a FLA-7000 phosphoimager (Fujifilm). Methylene blue was used to visualize rRNA.