Clc genomic workbench v5

Genomic DNA from the 22 environmental strains tested on HeLa cells was isolated as described above. The quantity was measured using both a Qubit 2.0 fluorometer with a Qubit dsDNA HS assay kit (Invitrogen, Thermo Fisher Scientific) and a 2200 TapeStation instrument with a genomic DNA ScreenTape assay (Agilent Technologies Inc., Santa Clara, CA, USA). Genomic libraries were prepared using a Nextera XT kit (Illumina Inc., San Diego, CA, USA) according to the manufacturer’s protocol and quantified by capillary electrophoresis, applying the Agilent High Sensitivity D5000 ScreenTape system (Agilent Technologies Inc.). Libraries were sequenced on a MiSeq machine (Illumina) using v2 reagents with 2 × 250-bp paired-end reads. Consequently, 81.6% to 98.8% of bases in sequencing reads had quality scores of 30 (Q30) or higher. De novo genome assembly was performed using CLC Genomic Workbench v5 (Qiagen GmbH). Rapid Annotation using Subsystem Technology (RAST server; https://rast.nmpdr.org/) was used for functional annotation of proteins (61 (link)).

Small RNAs were isolated and enriched from total RNA as described above, and ligated to a 5′ RNA adapter and a 3′ RNA adapter, as described previously^{57 (link)}. The ligation product was RT-PCR amplified and gel purified before sequencing on Illumina HiSeq 2000 platform. Three biological replicates were sequenced. Adapter sequences were first removed from raw sRNA reads. The resulting sRNA sequences were further processed to remove those containing low-complexity and t/rRNA sequences, and having lengths <15 bp or >29 bp. The remaining high-quality sRNA reads were aligned to the peach genome 1.0 and the 65-kb DAM sequence with perfect matches and reads with multiple alignments in the genome were excluded from further analysis. Raw read counts for each sRNA were normalized to RPKM and statistical analysis of changes of all sRNAs along DAM region (Sr) during temperature-dependent dormancy release and flowering was performed, using CLC Genomic Workbench V.5 (Qiagen, Hilden, Germany).

Genomic DNA was isolated using the QIAamp DNA Micro Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s procedure with a protocol for Gram-negative bacteria. Final elution was performed with nuclease-free water. DNA quality was assessed using a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, USA). The quantity was measured using both the Qubit 2.0 Fluorometer with Qubit dSDNA HS Assay Kit (Invitrogen, Thermo Fisher Scientific, Wilmington, USA) and the 2200 TapeStation Instrument with Genomic DNA ScreenTape Assay (Agilent Technologies Inc., St Clara, CA, USA). Libraries were prepared using the Nextera XT kit (Illumina Inc., San Diego, CA, USA) according to the manufacturer’s protocol and quantified by capillary electrophoresis applying the Agilent High Sensitivity D5000 ScreenTape System (Agilent Technologies Inc.). Libraries were sequenced on the MiSeq machine (Illumina) using v2 reagents with 2 × 250 bp paired-end reads. Consequently, 90.2 and 82.4% of bases of sequencing reads had quality scores ≥ Q30 for K1609 and K670, respectively. De novo genome assembly was performed using CLC Genomic Workbench v5 (Qiagen). Plasmid DNA was isolated using AccuPrep Plasmid Mini Extraction Kit (Bioneer Company, Daejeon, South Korea) according to the manufacturer’s procedure.