Sequel binding kit 3

Bacterial pellets were resuspended in 200 µL of 1× PBS and transferred to a 2-mL bead beating tube (Matrix E; MP Biomedicals). Proteinase K (20 µL; Qiagen) was added, and the cells homogenized (SPEX 1600 MiniG; 1 min; 1,500 Hz Fisher Scientific). DNA was extracted using the Qiagen DNeasy Blood & Tissue Kit according to the manufacturer’s instructions (Qiagen). The DNA was quantified using the 1× dsDNA HS kit (ThermoFisher Scientific on a Qubit). DNA was prepped with the SMRTbell Template Prep Kit 2.0 (Pacific Biosciences) to make PacBio SMRTbell libraries with barcodes sourced from the Barcoded Overhang Adaptor Kit 8A and 8B (Pacific Biosciences). The sequencing primers were annealed and bound to Polymerase 3.0 using the Sequel Binding Kit 3.0 (Pacific Biosciences). The bound complex was purified and sequenced on a PacBio Sequel I using an SMRT Cell M1 v3 tray (Pacific Biosciences). The spike-in controls for each PacBio Sequel I run were from the Internal Control Kit 3.0 (Pacific Biosciences).

The library preparation for sequencing on RSII system was performed as described before^{26 (link)}. For the sequencing on the Sequel platform, about 1.5 µg cDNA was treated with NEBNext End Repair Module (E6050, New England Biolabs) at room temperature for 5 min, followed by purification with Monarch PCR & DNA Cleanup Kit (T1030, New England Biolabs). The end-repaired cDNA was ligated with 2 µL barcoded adaptor (100-466-000, Pacific Biosciences) with T4 DNA Ligase (M0202, New England Biolabs) in 50 µL reaction volume at room temperature for 1 h, followed by purification with Monarch PCR & DNA Cleanup Kit (T1030, New England Biolabs). The un-ligated adaptor and cDNA were digested with E. coli Exonuclease III (M0206, New England Biolabs) and Exonuclease VII (M0379, New England Biolabs) in 1X standard Taq buffer at 37 °C for 1 h, followed by cleaning up with Monarch PCR & DNA Cleanup Kit (T1030, New England Biolabs). The ligated DNA was repaired with PreCR Repair Mix (M0309, New England Biolabs) at 37 °C for 30 min. The libraries were purified with 0.6X volume of AMPure PB beads (100-265-900, Pacific Biosciences) and pooled for sequencing runs. SMRT Link was used to generate the protocol for primer annealing, polymerase binding [Sequel Binding Kit 3.0 (101-613-900, Pacific Biosciences)], cleanup and final loading to SMRT Cells LR and sequencing using Sequel system.

SMRT bell libraries were prepared using the ‘Express Template Prep Kit 1.0’ protocol (Pacific Biosciences). A total of 5 µg gDNA was sheared to ∼ 15 kb fragments using g-TUBE (Catalog No. 520079, Covaris) plus centrifugation (2000 g, 2 min, twice). The fragments ware size-selected for 10 kb using the BluePippin system (Sage Science) by marker (0.75% DF Marker S1 High-Pass 6-10 kb vs3) for the 10–20 kb DNA target fragments. Quality control of the libraries was performed by Qubit fluorometer (Life Technologies, Carlsbad, CA) and Bioanalyzer 2100 (Agilent Technologies). The prepared library was loaded into SMRT cell 1M by Sequel Binding Kit 3.0 (Pacific Biosciences) and finally sequenced by CCS mode with the Sequel System (Pacific Biosciences).

To aid in genome annotation, we generated ISO-seq data for mixed RNA of five different tissues of Mo17, including root, silk, tassel and bract collected in the silking stage, and 14-d seedlings. All five tissues were frozen in liquid nitrogen after collecting and stored at −80°C before processing. Total RNA of each sample was extracted with TRIzol according to the manufacturer’s instructions. The integrity of the RNA was determined with the Agilent 2100 Bioanalyzed (Agilent Technologies) and agarose gel electrophoresis. The purity and concentration of the RNA were determined with the Nanodrop (Thermo Fisher Scientific) and Qubit (Thermo Fisher Scientific). Only high-quality RNA (RNA integrity number ≥ 8, OD260/280 = 1.8–2.2, OD260/230 ≥ 2.0) was used for library construction. Equal amounts of RNA from the five tissues were pooled together. Mixed RNA was reverse transcribed into cDNA using a SMARTer PCR cDNA Synthesis Kit (PacBio Biosciences). Double-stranded cDNA was then generated by PCR (PrimeSTAR GXL DNA polymerase). Resulted double-stranded cDNAs were DNA damage repaired, end repaired and ligated to sequencing adapters using SMRTbell Template Prep Kit 1.0 (Pacific Biosciences). The SMRTbell template was annealed to sequencing primer and bound to polymerase and sequenced on the PacBio Sequel platform using Sequel Binding Kit 3.0 (Pacific Biosciences).

Single molecule real-time (SMRT) sequencing was performed to identify modified bases of S. xylosus and genetically modified E. coli strains (Clark et al., 2012 (link)). DNA isolation was performed using the E.Z.N.A. Bacterial DNA-kit (Omega Bio-tek) according to the manufacturers instruction, except that lysostaphin (0.5 mg/ml) was included into the lysis buffer of the staphylococcal samples to weaken the cell wall. Library construction and sequencing (PacBio RS II) of S. xylosus followed the protocol described by Schiffer et al. (2019) (link). E. coli sequencing was performed on a PacBio Sequel instrument (SMRT cell 1M), partly at the functional genomics center Zurich (ETH Zürich), and partly at the research unit for environmental genomics Munich (Helmholtz Zentrum München). Therefore, the Sequel^® Binding Kit 3.0 (Pacific Biosciences of California) was used and the libraries were size-selected to around 6 to 7 kb. SMRT Analysis version 7.0 (Pacific Biosciences) was used for assembly (HGAP4), base modification and motif analysis of S. xylosus, SMRT Link version 10.1 for assembly, base modification and motif analysis of E. coli. For S. xylosus the assembled genomes were used as their own reference, while for E. coli, the assembly of strain DH10B available on NCBI (NC_010473) was used as a reference.