Rpn203b

Cells were lysed in lysis buffer (100 mM NaCl, 10 mM Tris pH 8.0, 25 mM EDTA pH 8.0, 0,5% SDS, 50 μg/mL Proteinase K) overnight at 37°C. Nucleic acids were extracted using standard phenol-chloroform extraction protocol and resuspended in DNase/RNase-free water. Nucleic acids were then fragmented using a restriction enzyme cocktail (20 U each of EcoRI, BamHI, HindIII, BsrgI, and XhoI). Half of the sample was digested with 40 U RNase H (MB085, NZYTech) for 48 hr at 37°C to be used as a negative control in R-loops blotting. Digested nucleic acids were cleaned with standard phenol-chloroform extraction and resuspended in DNase/RNase-free water. Nucleic acids samples were quantified in a NanoDrop 2000 spectrophotometer (Thermo Scientific), and equal amounts of DNA were deposited into a positively charged nylon membrane (RPN203B, GE Healthcare). Membranes were UV-crosslinked using UV Stratalinker 2400 (Stratagene), blocked in 5% (m/v) milk in PBSt (PBS 1× containing 0.05% [v/v] Tween 20) for 1 hr at room temperature, and immunoblotted with specific antibodies. For the loading control, membranes were stripped in 0.5% SDS for 1 hr at 60°C, followed by blocking and re-probing. Details of the antibodies used are included in Supplementary file 2C.

Total RNA isolated from the sucrose gradient fractions was resolved using a 10% polyacrylamide gel in 0.5x TBE buffer buffer (1x TBE buffer contains 89 mM Tris, 89 mM boric acid, and 2 mM EDTA) and electroblotted onto a nylon (N+) membrane (GE Healthcare RPN203B) at 20 V for 90 min at 4°C in 0.5x TBE buffer. The membrane was crosslinked and pre-hybridized in UltraHyb Hybridization Solution (Thermo AM8670) at 42°C for 1 hour, then incubated overnight with 50 pmol Met-tRNA_i specific probe (5′-TGGTAGCAGAGGATGGTTTCGAT-3′). The probe was labeled on the 5′ end with [γ- 32P] ATP (Perkin Elmer) using T4 polynucleotide kinase (NEB M0201) according to the manufacturer’s protocol. Membranes were washed twice by 20 mL 6x SSC for 5 min at 42°C and twice by 20 mL 2x SSC and twice by 20 mL 1x SSC (20x SSC contains 0.3 M sodium citrate in 3 M NaCl). Membranes were then wrapped in saran wrap, exposed to a phosphor screen overnight, and visualized by phoshor-imaging.

3 to 5 µg RNAs were separated on a 1.5% agarose gel and transferred overnight by capillarity on a nylon membrane (GE Healthcare, #RPN203B) in SSC 10X Buffer. RNAs were then UV-crosslinked to the membrane using a Stratalinker apparatus. Generation and incubation of the membrane with RNA dig-labeled probes was performed using the Dig-Northern-Starter Kit (Roche, #12039672910), following manufacturer’s instructions. Membranes were washed twice in 2X SSC 0.1% SDS and once in 1X SSC 0.1% SDS, for 10 min at 65 °C. Revelation was done according to the kit instructions using a Vilber Lourmat Fusion Fx7 detection device. Oligonucleotides used to generate DNA templates for RNA probes are listed in Supplementary Table 5.