Exonuclease 7

Each strain was growth in RB with the appropriate antibiotics (see "Growth media" above) overnight at 37°C with 250 rpm agitation.
gDNA was extracted with the Monarch Genomic DNA purification kit (New England Biolabs; Ipswich, MA, USA) from 1ml of culture.
Libraries from these genomic DNAs were sequenced using the PacBio RSII or Sequel I sequencing platform. Briefly for RSII, SMRTbell libraries were constructed from genomic DNA samples sheared to between 10 and 20 kb using the G-tubes protocol (Covaris; Woburn, MA, USA), end repaired, and ligated to PacBio hairpin adapters. Incompletely formed SMRTbell templates and linear DNAs were digested with a combination of Exonuclease III and Exonuclease VII (New England Biolabs; Ipswich, MA, USA). The SMRTbell library was prepared according to PacBio sample preparation protocol sequenced with C4-P6 chemistry with a 300 min collection time.
For Sequel I libraries, SMRTbell libraries were constructed from genomic DNA samples following the PacBio protocol for Sequel using the kit 100-938-900. DNA qualification and quantification were performed using the Qubit fluorimeter (Invitrogen, Eugene, OR) and 2100 Bioanalyzer (Agilent Technology, Santa Clara, CA). The libraries were prepared for binding following the PacBio guidelines generated by SMRT Link and run on a Sequel I machine.

SMRTbell libraries were prepared using a modified PacBio protocol adapted for NEB reagents. Genomic DNA samples were sheared to an average size of ~ 6–10 kb using the G-tube protocol (Covaris; Woburn, MA, USA), treated with FFPE, end repaired, and ligated with hairpin adapters. Incompletely formed SMRTbell templates were removed by digestion with a combination of exonuclease III and exonuclease VII (New England Biolabs; Ipswich, MA, USA). The qualification and quantification of the SMRTbell libraries were made on a Qubit fluorimeter (Invitrogen, Eugene, OR) and a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). SMRT sequencing was performed using a PacBio RSII (Pacific Biosciences; Menlo Park, CA, USA) based on standard protocols for 6–10 kb SMRTbell library inserts. Sequencing reads were collected and processed using the SMRT Analysis pipeline from Pacific Biosciences (http://www.pacbiodevnet.com/SMRT-Analysis/Software/SMRT-Pipe) [53 (link)]. Next-generation SMRT sequencing technology from Pacific Biosciences Inc. allowed the assembly of a complete circular genome. It also enabled the determination of the m6A and m4C modified motifs. [54 (link)–56 (link)].

After landing, samples were removed from the plane, immediately frozen at −20°C and returned to the laboratory where they remained frozen until sample post-flight processing. Prior to sequencing library preparation, all samples were digested with Exonuclease VII (New England Biolabs) to remove ssDNA tails from the partially polymerized DNA species in order to yield trimmed dsDNA molecules containing only polymerized DNA. Following this, the NEBNext Ultra II Library Preparation kit (New England Biolabs) was used to prepare samples for sequencing on the NovaSeq 6,000™ (Illumina, San Diego, CA). Further information regarding the post-flight processing protocol is documented in Supplementary Materials.

To digest the unincorporated primer pools in the completed PCR reactions prior to capillary electrophoresis, 5 μL of PCR product was transferred to wells of a 96-well PCR plate and 1 μL of either Exonuclease I (20U/μL)(NEB) or Exonuclease VII (10U/μL)(NEB) were added. The plate was incubated at 37°C for 30 minutes followed by a heat inactivation step of 95°C for 10 minutes. The samples were then analyzed by capillary electrophoresis as described above.

Total RNA was extracted with TRIzol (Fisher #15596018), following the manufacturer’s instructions, for intact infiltrated leaves as well as leaf protoplasts isolated using the above mentioned method for scRNA-seq. DNase treatments were performed with RQ1 RNase-Free DNase (Promega #PR-M6101). Three biological replicates were performed for samples of Pst DC3000- or mock-infiltrated leaves, and one repeat was made for leaf protoplasts of each sample. cDNA libraries were prepared with QuantSeq FWD kit (Lexogen), according to the manufacturer’s protocol. The fragment size distribution was evaluated by a Bioanalyzer 2100 (Agilent). The library pool was treated using Exonuclease VII (NEB), SPRI-bead purified with KapaPure beads (Kapa Biosystems /Roche), quantified via qPCR with a Kapa Library Quant kit (Kapa Biosystems) on a QuantStudio 5 RT-PCR system (Applied Biosystems). Sequencing was performed at the University of California Davis Genome Center using a HiSeq 4000 (Illumina) platform with single-end 100 bp reads.