Sequel system

The male and female samples from D. magna were prepared for long-read isoform sequencing (Iso-Seq). Three biological replicates for each sample were conducted. The full-length cDNA was produced from 300 ng of total RNA for each sample using the NEBNext Single Cell/Low Input cDNA Synthesis & Amplification Module (New England Biolabs, MA USA) and Iso-Seq Express Oligo kit (Pacific Biosciences, CA USA) according to manufacturer’s protocols. Additionally, ribosomal RNA (rRNA) was removed from 12 μg of total RNA of each sample using the Ribo-Zero Gold kit (Illumina, CA USA). The rRNA-depleted RNA samples were then polyadenylated using the SMARTer smRNA-Seq kit for Illumina (Clontech Laboratories, Inc., CA USA). The full-length cDNA was generated following the same protocols as those used for total RNA. After size selection with the ProNex beads (Promega, WI USA), the full-length cDNA libraries were constructed with the SMRTbell Express kit 2.0 (Pacific Biosciences, CA USA). The concentration and the size distribution of each library were measured using the Qubit 4 Fluorometer (Thermo Fisher Scientific, MA, USA) and Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA), respectively. Each library was then sequenced on the Sequel system (Pacific Biosciences, CA USA) using one SMRT Cell 1 M v3 LR with Binding Kit 3.0 and Sequencing Kit 3.0, with a collection time of 20 h.

To determine the methylome of B. cenocepacia, gDNA extracts were analyzed with single-molecule real-time (SMRT) sequencing technology. gDNA samples of both wild-type and mutant strains were run on a Pacific Biosciences Sequel System (250× coverage) according to the manufacturer’s guidelines. Library preparations were multiplexed as data output of approximately 2 Gb per genome was expected, and a single SMRT Sequel cell provides up to 6 Gb data. Initial data output was processed with SMRT Link software (Pacific Biosciences). Identification of the modified bases and analysis of the methylated motifs were performed with the Base Modification and Motif Analysis application (SMRT Link v6.0; Pacific Biosciences). In-depth data analysis was performed with CLC Workbench Genomics (v11.0.1; Qiagen). Differential analysis between wild type and mutants was performed to identify methylation motifs specifically associated with certain DNA MTases. Previously predicted promoter regions and transcription start sites of B. cenocepacia were used to determine the methylation profile of regulatory regions (64 (link)). Virtual Footprint software (promoter analysis mode, default search parameters) was used to assess similarity of the methylation motifs to known TF binding sites (65 (link)).

Wild type S. fungicidicus as well as strains F1, F2, F7, F8, F9 and F10, were subject to whole genome sequencing. gDNA was isolated using NEB Monarch® Genomic DNA Purification Kit. The gDNA was then sheared using a gTube (Covaris, roughly 10 kb fragments) and prepared for sequencing following the SMRTbell Express Template Preparation Kit 2.0 protocol. Sequencing data were obtained using the Sequel system (Pacific Biosciences) with a 10 h acquisition. Assembly of the genome was performed using the HGAP4 algorithm in SMRT Link v8.0. Assembled genomes were aligned using mauve multiple genome alignment with Geneious Prime 2021.1.