Nylon n membrane

Each repeat-containing strain transformed with its cognate sgRNA and nucleases was grown for 4 h in 2% galactose SC-URA-LEU. Total RNAs were extracted using standard phenol–chloroform procedure (23) or the miRVANA kit, used to extract very low levels of small RNAs with high efficacy (ThermoFisher). Total RNA samples were loaded on 50% urea 10% polyacrylamide gels and run at 20 W for 1 h. Gels were electroblotted on N⁺ nylon membranes (GE Healthcare), hybridized at 42°C using a SpCas9, SaCas9, FnCpf1 or SNR44 oligonucleotidic probe. Each probe was terminally labeled with γ-³²P ATP in the presence of polynucleotide kinase, purified on a Sephadex G25 column (MicroSpin G25 column, GE Healthcare) and its specific activity was verified to be at least 1.2×10⁸ cpm/μg, and denatured (5′ at 95°C) before hybridization.

tRNAs were extracted from yeast cultures using standard methods (35 (link)) with some modifications (36 (link)). tRNA samples were resuspended in sodium acetate buffer (pH 4.6) and stored at –80°C.
Northern blot analysis was carried out by separating 5 μg tRNA per lane on a 25 cm-long 8 M urea, 0.1 M sodium acetate (pH 5) denaturing acrylamide gel (10%: 19:1 acrylamide:bisacrylamide). Gels were run at 80 mA for 36 h at 4°C with 7-hourly buffer re-circulation and semi-dry blotted onto GE-Healthcare Nylon N+ membrane using 1× tris borate–EDTA (TBE) buffer at constant current (1 mA/cm²) for 1 h at 4°C. tRNAs were crosslinked using UV (120 000 μJ/cm²), before probing using 3′ end biotin-labelled oligonucleotide probes; tRNA^Gln_UUG, tRNA^Lys_CUU and tRNA^Arg_UCU were detected using primers A6, A7 and A17 respectively (Supplementary Table S1). Probe labelling was carried out using the Pierce Biotin 3′ End DNA Labelling Kit. Probing of the blot was performed using the North2South™ Chemiluminescent Hybridization and Detection Kit (Thermo-Fisher) according to the manufacturer's instructions. Probe-tRNA hybridizations were carried out overnight at 42°C, except the tRNA^Arg_UCU probe hybridization, which was carried out at 70°C, reduced in 10°C increments per hour to 40°C after overnight incubation, before then washing and detection.

Total RNA was extracted from plant tissue using the TRIzol reagent (Invitrogen). The extracted RNA was separated on a 1.2% agarose gel containing formaldehyde, transferred to a nylon N⁺-membrane (GE Healthcare), and cross-linked with ultraviolet light. The membrane was stained with a methylene blue solution (0.5 N NaAc, pH5.2 and 0.04% methylene blue) and rinsed with distilled water to produce clearly visible bands, which were used as the loading control. The DNA fragments corresponding to position 75–435 of the Fd I mRNA sequence were labeled with [α-³²P]-dCTP using the Rediprime II DNA Labeling System (GE Healthcare). Signals were visualized on exposed film.

Mock- and p150CAF-1-depleted Xenopus high-speed egg extract (HSE) were prepared as previoulsy (Ray-Gallet and Almouzni, 2004 (link)). Nucleosome assembly was performed on pBS plasmid damaged by UV (500 J/m2) to promote DNA synthesis as previously described (Ray-Gallet and Almouzni, 2004 (link)) except that the reaction mixed contained 3.2 µM of biotin-14-dCTP (Invitrogen, Ref 19518–0189) instead of [α³²P]-dCTP. The p150CAF-1-depleted extracts were complemented with 50 ng of isolated/reconstituted SpCAF-1 complex composed of WT or mutated Pcf1. After DNA purification, samples were by processed for gel electrophoresis (1% agarose) to resolve topoisomers as previously described (Ray-Gallet and Almouzni, 2004 (link)). After staining with Ethidium bromide to visualize total DNA and gel transfer on a Nylon N+membrane (GE Healthcare Ref RPN203B) (Qbiogen) for 45 min at 40 mbar in 10 x SSC, the membrane was rinsed in PBS, air dried and DNA was crosslinked to the membrane using Stratalinker (Bio-Rad). DNA synthesis was visualized by detecting biotin with the Phototope-Star detection kit (New England Biolabs Ref N7020S) and images acquired on a Chemidoc system (Bio-Rad).