Dnase 1 buffer

Nuclei from 200 adult flies were extracted (Wilson et al., 1995) and the resulting nuclear suspensions were separated into mock‐ or DNase I‐treated groups. 10 µL DNase I (2000U/mL) and 10 µL 10x DNase I buffer (New England BioLabs, M0303S and B0303S) were added to the DNase I‐treated nuclear suspension in a final volume of 100 µL, and only 10x DNase buffer was added to mock‐treated groups. Reactions were incubated in a 37°C water bath for 10 – 15 min. The subsequent DNA cleaning and library preparations steps are described in (Follows et al., 2007). DNA from mock‐treated samples was purified and used as control for final normalization analysis.

APOBEC-YTH and APOBEC-YTH^mut were purified and in vitro DART-Sanger sequencing assays were performed as previously described (Tegowski et al., 2022 (link)) with minor modifications. Briefly, total RNA was isolated from adult heads with TRIzol (Invitrogen) and treated with DNase I (NEB). RNA was isolated once more with TRIzol (Invitrogen) to remove DNase I and DNase I Buffer (NEB). Next, 200 ng of purified RNA from Drosophila heads was incubated with 1000 ng of purified DART protein in DART buffer (10 mM Tris-HCl, pH 7.4, 50 mM KCl, 0.1 M ZnCl₂) and 1 µL of RNaseOUT (Invitrogen) in a total volume of 200 µL for 4 hr at 37°C. RNA was isolated with the QIAGEN Plus Micro Kit (QIAGEN) and stored at –80°C before being thawed for downstream Sanger sequencing analysis. cDNA was made using iScript Reverse Transcription Supermix (Bio-Rad). PCR amplification of Sxl pre-mRNA was carried out with Phusion High Fidelity PCR Kit (NEB). The resulting PCR product was PCR-purified using the QIAGEN PCR Purification Kit (QIAGEN). Samples were submitted for Sanger sequencing (McLabs) and %C-to-U editing was quantified using EditR software (Kluesner et al., 2018 (link)).

To demonstrate that only the full-length genes contain the anti-sense oligo of the promoter recognizable by the T7 RNA polymerase, 5 μL of the product from Step 3 was used directly for mRNA transcription reaction (20 μL total volume) using the TranscriptAid T7 High Yield Transcription kit (Thermo Fisher). After a 1.5-hour incubation at 37°C, RNase-Free DNase I (2 U, NEB) and DNase I Buffer (NEB) were added to the mixture to remove the DNA template (50 μL final reaction volume). The transcription products were cleaned up using RNA Clean & Concentrator-25 kit (Zymo Research; Irvine, CA). An aliquot of the synthesized mRNA was visualized on denaturing agarose gels (1.5% agarose and 1% bleach [21 (link)]) stained with EtBr. RiboRuler RNA Ladder, High Range (Thermo Scientific) was used for size comparison.

Random, optimized primers and probes were designed using the genome of Loxodonta africana for the detection of pEA-CRR1Φ of pEA-CRR2Φ to ensure no cross-reactivity with E. amylovora (Table 3). Previously developed primers and probes were used for Ea6-4 and ΦEa21-4 (30). In this system, a plasmid containing the Ea6-4 and ΦEa21-4 PCR amplicons was diluted to 10¹¹, 10⁸, and 10⁵ copies/mL to create a standard curve for quantification (30 (link)). qPCRs contained 2 µL of sample, 200 nM each primer, and 100 nM each probe in EVOlution Probe qPCR mix (Montreal Biotech Inc., Montreal, QC, Canada). Reactions were performed in a qTOWER G3 (Analytik Jena, Jena, Germany) or a Stratagene Mx3005P (Agilent Technologies, Santa Clara, CA, USA) qPCR thermocycler under the following conditions: 15 min at 95°C followed by 40 cycles of 15 s at 95°C and 45 s at 54°C. Prior to the quantification of ΦEa21-4, phage samples were treated with DNase to removed non-encapsidated phage genomes as previously described (31 (link)). Briefly, an 8 µL sample of phage was combined with 1 µL of 10x DNase I buffer (B0303D, NEB, Ipswich, MA, USA) and 1 µL DNase I (M0303S, NEB, Ipswich, MA, USA) in a 96-well plate. The samples were then incubated for 40 min at 37°C, followed by 20 min at 95°C, and a hold at 4°C. Phage were also quantified through plaque assays using a soft agar overlay (27 , 37 ).

TS600 and TS622 were grown in triplicate to an optical density (measured at 600 nm) of ∼0.5 in liquid ASW-YT-S and used to inoculate (1:100) 300 ml of ASW-YT-Pyr per strain and allowed to grow to an optical density (measured at 600 nm) of ∼0.05 to encourage chromatin reprograming. Cultures were rapidly chilled and pelleted at 8,000 × g for 5 min and then resuspended in 1.0 mL of Trizol (Invitrogen) with a 10-min incubation at room temperature. A total of 200 μl of chloroform was added followed by centrifugation at 10,000 × g at 4°C for 15 min yielding an RNA-containing aqueous layer which was added to 500 μl of isopropanol and incubated at room temperature for 10 min. Centrifugation at 10,000 × g for 15 min at 4°C produced an RNA pellet that was washed with 1 ml of 75% ethanol and subsequently resuspended in 88 μl of RNase-free H₂O, 10 μl of DNaseI buffer, and 1 μl of DNaseI (New England Biolabs) to digest residual DNA (37°C for 30 min). Replicate samples were prepared identically.

Dissected hypothalamus tissue was homogenized in lysis buffer (10mM Tris HCl [pH 7.4], 0.5% SDS, 100mM EDTA, 300μg/ml proteinase K) and incubated at 50°C for 2 hours. Total nucleic acid was recovered from the completely lysed sample by ethanol precipitation in 2 volumes of 100% ethanol at room temperature (for 30 minutes), and pelleting by centrifugation. The pellet was washed once in 2 volumes of 70% ethanol, and the nucleic acid pellet was resuspended in hydrolysis buffer containing 1x DNase I buffer (NEB), 1mM zinc sulphate, DNase I (NEB) and Nuclease P1 (Sigma). After 4 hours, the sample was mixed thoroughly and digested for a further 8 hours. After 12 hours at 37°C, the sample was heated to 92°C for 3 minutes and cooled on ice. Two volumes of 30mM sodium acetate, 1mM zinc sulphate [pH 5.2] were added plus additional Nuclease P1 and the nucleic acids were further digested to deoxyribonucleotide and ribonucleotide 5’ monophosphates for a further 24 hours at 37°C. The samples were then subjected to HPLC as set out below.