Rnase h

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.

Half of each in vitro transcription product was treated with 10 U RNase H (MB085, NZYTech) at 37°C overnight to serve as negative control. Then, all samples were treated with 0.05 U RNase A (10109142001, Roche) at 350 mM salt concentration for 15 min at 37°C and ran on agarose gel. Nucleic acids were transferred overnight to a nylon membrane through capillary transfer. The membrane was then UV-crosslinked twice, blocked in 5% milk in PBSt for 1 hr at room temperature, and incubated with the primary antibody at 4°C overnight. Signal quantification was performed using ImageJ. Details of the antibodies used are included in Supplementary file 2C.

The cDNA was synthesized from 100 ng of RNA using an NZY M-MuLV First-Strand cDNA Synthesis Kit (NZYtech, Lisbon, Portugal) for a final volume of 10 µL. Samples were incubated in DNA Engine^® Thermal Cycler (Bio-Rad Laboratories, Hercules, CA, USA) at 25 °C for 10 min, 37 °C for 50 min, and 85 °C for 5 min. Afterwards, 0.5 µL of RNase H (NZYtech, Lisbon, Portugal) was added and samples were stirred in a vortex and incubated in a heat block at 37 °C for 20 min. The synthetized cDNA was stored at −20 °C for further analysis.