S9.6 antibody

DNA-RNA immunoprecipitation (DRIP) was performed as described in Ariel et al. [52 (link)]. Non-crosslinked seedlings were used for nuclei purification, and samples were sonicated using a water bath Bioruptor Pico (Diagenode; 30 s on/ 30 s off pulses, at high intensity for 4 cycles). Chromatin samples were incubated with 50 ml of washed Protein G Dynabeads pre-coated with 1 mg of S9.6 antibody (Millipore MABE1095) for 16 h at 4 °C. Samples treated with RNAseH (Invitrogen) for 2 h at 37 °C were used for DRIP in parallel as a negative control. After washing, DNA was recovered for qPCR over R-loop-related loci (primers used are listed in Additional file 2: Table S4).

The following DRIB protocol was followed to detect R-loops (47 (link), 48 (link)). The nucleus was isolated by hypotonic homogenization. Next, the nucleus was lysed by lysis buffer (1% SDS, 10 mM Tris-HCl pH 7.5, 1 mM EDTA, 100 mM NaCl, 50 μg/ml Proteinase K) at 55 °C for 2 h. This was followed by phenol: chloroform extraction and 20 μg/ml RNaseA (sigma) treatment for 30 min at 37 °C. The genomic DNA prepared was sonicated in Tris-HCl pH 7.5 with 100 mM NaCl buffer in a probe sonicator thrice for 30 s each in 20% amplitude to yield a maximum fragment size of around ∼1000 bps. For further nucleic acid fragmentation a cocktail of restriction enzymes and purified by phenol: chloroform extraction. This fragmented nucleic acid was transferred to Hybond N membrane (Merck) using a slot blot system and DNA: RNA hybrid was detected by S9.6 antibody (Merck). dsDNA was detected using anti-dsDNA antibody (abcam). Digestion of DNA: RNA hybrid was carried out using RNaseH (NEB).

Genomic DNA was isolated from 2.5 × 10⁷ cells using the High Pure PCR template preparation kit (Roche). A 1.1-μg amount of DNA was then either treated with 10 U recombinant E. coli RNase H (NEB) or double-distilled water (ddH₂O) in 1× RNase H buffer (2 h, 37°C). All samples were further incubated with 10 μg RNase A (Thermo Fisher) (1 h, 37°C) in a final concentration of 500 mM NaCl. The DNA samples were spotted on a Hybond N+ membrane (GE Healthcare) using a dot blot apparatus in a 2-fold serial reduction and cross-linked with UV (0.12 J). To quantify R-loop formation, the membrane was blocked in 5% milk/PBS (overnight, 4°C). A 1-μg/ml concentration of S9.6 antibody (Merck Millipore) in 0.1% Tween/PBS was then incubated with the membrane (1 h, RT). After three washes with 50 ml 0.2% Tween/PBS, the membrane was incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody (Dianova) (1:20,000) (1 h, RT). After three washes with 50 ml 0.2% Tween/PBS, the HRP signal was developed with Western Lightning Plus-ECL solutions (PerkinElmer) and imaged using an iBright imaging system (Thermo Fisher). iBright Analysis software was used for quantification of signals.

One microgram of chromatin-associated, phenol/chloroform-isolated NA were either treated with RNase H (Roche) or untreated for 2 h at 4°C. This material was then incubated with 25 μl of magnetic Protein G Dynabeads (Thermo Fisher Scientific) that had been coupled to 1 μg of the monoclonal S9.6 antibody (Merck Millipore, cat. no. MABE 1095) O/N at 4°C in a rotating wheel. The next day, the bead-protein-NA complexes were washed three times for 5 min at RT with ChIP-wash buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA pH 8.0, 150 mM NaCl, 20 mM Tris–HCl pH 8.0 and protease inhibitors (Roche)) and once for 10 min at RT with the ChIP-final wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA pH 8.0, 500 mM NaCl, 20 mM Tris–HCl pH 8.0, and protease inhibitors (Roche)). The bead-protein-NA complexes were then resuspended in 100 μl of RDIP-elution buffer and processed for detection of MSR and LINE L1MdA 5′UTR RNA:DNA hybrids by RNA amplification as described above.

The following protocol was carried out for immunofluorescence. Leishmania cells were washed twice with 1× PBS (phosphate buffer saline) and seeded in poly-lysine coated 10 well chamber slides or coverslips for 1 h to properly adhere cells at room temperature. The unadhered cells were washed with 1× PBS 4 to 5 times and fixed with 4% paraformaldehyde (Sigma) for 30 s and again washed with 1× PBS 4 to 5 times. The fixed cells were treated with 0.5% Triton X 100 (Sigma) for 15 min and subsequently washed, and incubated with 5% BSA (Sigma)in 1× PBS (blocking solution) for 1 h at room temperature. The blocking solution was removed and cells were incubated with the desired primary antibody for the incubation period as standardized for each antibody. This was followed by several washes and, incubation with required Alexa fluor 488 or Alexa 568 conjugated secondary antibodies (Invitrogen) for 1 h at room temperature. The cells were washed and stained with nuclear dye 1× DRAQ5 (CST) for 10 min. The cells were washed 6 to 7 times to remove excess stain followed by drying and mounting with anti-fade gold mounting medium (Invitrogen). For curing the slides were kept 24 h at room temperature and high-resolution imaging was done in a confocal laser scanning microscope (Olympus FV3000). DNA–RNA hybrid and dsDNA were detected by S9.6 antibody (Merck) and dsDNA antibody (Abcam), respectively.