Qubit 4.0 fluorimeter

Developing fruits of the red raspberry cultivar ‘Anitra’ were collected at three maturity stages; unripe, turning, and fully mature. Fruit samples from each maturity stage were collected at Graminor Njøs (Norway), where they were divided into three biological replicates, snap frozen in liquid nitrogen and stored at -80°C until the samples were processed further. Tissue samples were ground to a fine powder under liquid nitrogen, and total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen, Germany) following the manufacturer’s instructions. The concentration and purity of the resultant RNA was measured using a QIAxpert spectrophotometer (Qiagen, Germany) and the integrity of the RNA was determined using a Qubit 4.0 fluorimeter (Thermo Fisher Scientific, UK). Samples with an RNA integrity number (RIN) value above 7.0 were submitted for subsequent RNA-Seq. Library preparations were performed using the NEB Next® ultra RNA Library Prep Kit (Biolabs, Inc., Beijing, China) and 150 bp paired-end sequencing was performed by Norwegian Sequencing Centre (Oslo University Hospital, Norway) using the HiSeq2500 platform (Illumina Inc., Beijing, China) to yield between 12.6 and 19.4 Gb of data per sample.

Genomic DNA was obtained from 15 bolting and 24 non-bolting individuals of the L14 line using a modified CTAB DNA extraction method (Doyle and Doyle, 1990 ). The quality of the extracted DNA was checked on a 0.8% agarose gel, and quantification of the same was done using a Qubit 4.0 fluorimeter (Thermo Fisher Scientific, Waltham, MA, Unites States). The DNA samples were subjected to a Restriction site-associated DNA technology (RAD-seq) with a HiSeq 2000 sequencing system using 150-bp paired-end sequencing (Illumina Inc., San Diego, CA, United States). Briefly, the extracted genomic DNA was digested using PstI (New England Biolabs, Ipswich, MA, United States) and ligated with Illumina RE-site compatible P1 adapters (Illumina Inc., San Diego, CA, United States). The size selection of 300–500 bp was performed using solid-phase reversible immobilization (SPRI) beads (AgencourtAMPure XP Beads) from Beckman Coulter (Indianapolis, IN, United States). The resulting fragments were end-repaired, 3′-adenylated, and ligated with universal P2 adapters. The libraries were diluted and pooled with an equimolar concentration of each library and sequenced on an Illumina HiSeq 2500 platform (Illumina, San Diego, CA, United States) following the recommendations of the manufacturer.

DNA isolation was performed from the studied samples using commercial DNA-Extran-2 kits (CJSC Syntol, Russia) according to the manufacturer’s recommendations. The concentrations of double-stranded DNA were measured on a Qubit 4.0 fluorimeter (Thermo Fisher Scientific (formerly Life Technologies), Wilmington, DE, USA). The DNA purity was checked by evaluating the absorption ratio of OD260/OD280 on a NanoDrop2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA).

Dual DNA and RNA isolation was performed from four formalin-fixed paraffin-embedded (FFPE) tissues using the Maxwell^® RSC DNA/RNA FFPE Kit with the Maxwell^® RSC Instrument. For each sample, areas were characterized by 100% tumor cells. Nucleic acid concentrations were determined by fluorometric quantitation using Qubit 4.0 Fluorimeter with Qubit dsDNA HS Assay Kit and Qubit RNA HS Assay Kit (Thermo Fisher Scientific, Inc.).

Small RNA libraries from serum samples were obtained using QIAseq^® miRNA Library kit (Qiagen, Hilden, Germany), according to the manufacturer protocol. NGS Library Quality Control (QC) analysis and quantification were performed before sequencing: a) High sensitivity DNA electrophoresis by LabChip GX Touch Nucleic Acid Analyzer (PerkinElmer, Massachusetts, USA) using HT DNA 5K/RNA LABCHIP kit (D-MARK Biosciences, Toronto, Canada) according to the manufacturer’s instructions. We obtained typical electropherograms from small RNA libraries that show a peak between 170-180 bp corresponding to miRNA-sized library; b) quantitative polymerase chain reaction (qPCR) according to the manufacturer’s protocol, using three different primers provided by QIAseq^® miRNA Library kit (Qiagen): the first, called NGS 3C Primer, for assessing the performance of 3’ adapter ligation; the second, NGS 5C Primer, for assessing the performance of 5’ adapter ligation and the third, NGS RTC Primer, for the performance of reverse transcription reaction. We obtained a value of threshold cycle (CT) less than 28 indicating all these steps were performed correctly. NGS library concentration was determined by Qubit dsDNA HS assay (Thermo Fisher Scientific) by Qubit^® 4.0 Fluorimeter, according to the manufacturer’s protocol.

Twenty-four mcr-positive isolates were characterized by WGS. Genomic DNA was extracted using DNeasy Blood & Tissue Kit and QIAcube Connect Device (QIAGEN, Hilden, Germany). For short-read sequencing, library preparation was performed using Illumina DNA prep kit (Illumina, San Diego, CA, USA) according to manufacturer instructions. Capillary electrophoresis using the Agilent 2100 Bioanalyzer system and Agilent High Sensitivity DNA Kit (Agilent, Santa Clara, CA, USA) was used for estimation of library quality. Concentration of the libraries was measured using the Qubit 4.0 fluorimeter with Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Sequencing was performed on the Illumina NextSeq 550 System with the NextSeq 500/550 Mid Output Kit v2.5 (300 Cycles) and on the Illumina MiSeq System with MiSeq Reagent Kit v2 (500-cycles) (Illumina, USA). For long-read sequencing, library preparation was performed with NEBNext Companion Module (New England Biolabs, Ipswich, MA, USA) and Ligation Sequencing Kit (Oxford Nanopore Technologies (ONT), Oxford, UK) in accordance with manufacturer’s protocol. Samples were multiplexed using Native Barcoding Expansion 1–12 (ONT, UK). Library concentration was measured with Qubit dsDNA HS Assay Kit and Qubit 4 Fluorometer (Thermo Fisher Scientific, USA). Sequencing was performed on the MinION platform and R9.4.1 Flow Cells (ONT, UK).

Leaves from plant materials listed in Table 1 were collected to extract DNA representing the material from SBSI. For the material from Strube Research, 70 plants from 5 extremely resistant lines (SR-R1, SR-R2, SR-R3, SR-R4, and SR-R5) and 110 plants from 5 extremely susceptible lines (SR-S1, SR-S2, SR-S3, SR-S4, and SR-S5) were selected for DNA extraction. Genomic DNA was obtained using a modified CTAB DNA extraction method [33 ]. The quality of the extracted DNA was checked on a 0.8% agarose gel and quantification of the same was done using a Qubit 4.0 fluorimeter (Thermo Fisher Scientific, Waltham, MA, USA). The DNA samples were subjected to a restriction site-associated DNA technology (RAD-seq) [34 (link)] with a HiSeq 2000 sequencing system using 150-bp single-end sequencing (Illumina Inc., San Diego, CA, USA).