Qubit 3.0 fluorometer

The quantity and quality of the purified total RNA was assessed using a Thermo Fisher Scientific Qubit 3.0 fluorometer with the Qubit RNA BR Assay Kit (Cat # Q10211, Thermo Fisher Scientific) and an Advanced Analytical Fragment Analyzer System using a Fragment Analyzer RNA Kit (Cat # DNF-471, Agilent Technologies, Santa Clara, USA), respectively. Sequencing libraries were constructed using an Illumina TruSeq Stranded mRNA Library Prep kit (20020595, Illumina, San Diego, USA) in combination with TruSeq RNA UD Indexes (Cat # 20022371, Illumina) according to Illumina’s guidelines. The cDNA libraries were evaluated using a Thermo Fisher Scientific Qubit 3.0 fluorometer with the Qubit dsDNA HS Assay Kit (Cat # Q32854, Thermo Fisher Scientific) and an Agilent Fragment Analyzer (Agilent) with an HS NGS Fragment Kit (Cat # DNF-474, Agilent), respectively. Pooled cDNA libraries were sequenced 100 bp single-end on one lane of an Illumina HiSeq 3000 instrument (Illumina). All base call files were demultiplexed and converted into FASTQ files using Illumina bcl2fastq conversion software. The quality control assessments, generation of libraries, and sequencing were conducted by the Next Generation Sequencing Platform, University of Bern.

Total RNA was isolated from the samples mentioned above using RNeasy Mini Kit with on-column DNase treatment using the RNAse-Free DNase Set (both Qiagen, Hilden, Germany), following manufacturer’s protocol. Quantification and quality control of isolated RNA were assessed with both Qubit 3.0 fluorometer (RNA HS assay kit, ThermoFisher, Massachusetts, MA, USA) and Agilent 2100 Bioanalyzer (Agilent RNA 6000 Pico Kit, Agilent Technologies, Palo Alto, CA, USA). Samples with both a final yield comprised between 0.03 – 1.25 ng/µl and a RIN of 9 were prepared for sequencing with the SMARTer Stranded Total RNA-Seq v2 – Pico Input Mammalian Kit (Takara Bio Inc., Shiga, Japan). Fully automated library preparation was performed on a Microlab Star Hamilton robotic station (Hamilton Company, Reno, NV, USA). Briefly, 8 µl per sample were used for the cDNA synthesis via the SMART^® technology (SMART technology, Clontech, USA). Thereafter, each sample was amplified to generate Illumina-compatible libraries according to the manufacturer’s guidance. Libraries were validated using the Agilent 2100 Bioanalyzer (Agilent High Sensitivity DNA Kit, Agilent Technologies, Palo Alto, CA) and the Qubit 3.0 fluorometer (DNA HS assay kit, ThermoFisher, Massachusetts, MA, USA). Libraries were paired-end sequenced on two SP flow cell on NovaSeq 6000 system (Illumina Inc., San Diego, CA, USA).

The Qubit 3.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, United States) was used to control the quality of the DNA, which was subsequently used to perform amplification of the V3–V4 hypervariable regions of prokaryotic16S rDNA. Pairing primers were designed by GENEWIZ (South Plainfield, NJ, United States). The sequence of the forward primer was “CCTACGGRRBGCASCAGKVRVGAAT,” and the reverse primer contained the sequence “GGACTACNVGGGTW TCTAATCC.” Each polymerase chain reaction volume was 25 μL, containing 2.5 μL of TransStart Buffer (TransGen, Beijing, China), 2 μL of dNTPs, 1 μL of each primer, and 20–30 ng of template DNA. Then, the indexed adapters were attached to the ends of the amplicons to generate indexed libraries for subsequent next-generation sequencing using the Illumina platform (San Diego, CA, United States). The libraries were validated using the Qubit3.0 Fluorometer (Thermo Fisher Scientific) and quantified to 10 nmol. The Illumina MiSeq instrument was used to load and sequence the DNA libraries according to the manufacturer’s instructions. Sequencing was performed under the paired-end 250-bp mode. The raw reads were trimmed using the Cutadapt software to generate clean reads.

For RNA and DNA extraction, 2–10 sections of 10 µm-thick formalin-fixed paraffin-embedded tissue were prepared. After macrodissection of the tumor area, RNA and DNA were extracted using the Recover All Total Nucleic Acid Isolation kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. RNA concentrations were determined with the Qubit RNA HS Assay Kit and Qubit 3.0 Fluorometer (Thermo Fisher Scientific). DNA concentrations were measured with the Qubit dsDNA HS Assay Kit and Qubit 3.0 Fluorometer (Thermo Fisher Scientific).

Optical Genome Mapping (OGM) was performed according to the manufacturer’s instructions. Briefly, DNA was isolated from 1.5 × 10⁶ frozen cells using the Bionano Prep SP Frozen Cell Pellet DNA Isolation Protocol v2 (#30398 Rev B, Bionano Genomics Inc., San Diego, CA, USA). The DNA concentration was determined with the Qubit^TM dsDNA BR Assay Kit and a Qubit 3.0 Fluorometer (Thermo Fisher Scientific). Subsequently, the DNA was labeled with Bionano Prep Direct Label and Stain Kit (Protocol #30206 Rev G; Bionano Genomics Inc.) and the concentration determined with the Qubit^TM dsDNA HS Assay Kit and a Qubit 3.0 Fluorometer (Thermo Fisher Scientific). The labeled DNA was loaded on a Saphyr G2.3 chip and imaged with the Saphyr device (Bionano Genomics Inc.). The data were analyzed and visualized on the Bionano Access Server with the de novo pipeline (Tools Version 1.7.1, reference genome GRCh38/hg38). The recommended filter settings were applied and the frequency of SVs in the Bionano control database was set to 0%.

RNA samples were quantitated using the Qubit HS RNA kit (Thermo Fisher Scientific) with a Qubit 3.0 Fluorometer (Thermo Fisher Scientific), following manufacturer’s instructions. The 260/280 and 260/230 ratios of absorbance values were used to assess the purity of RNA using a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific). A 260/280 ratio of ~ 2.0 and 260/230 ratio in the range of 2.0–2.2 was accepted as “pure” for RNA. Lower ratios may indicate the presence of protein, phenol, EDTA, carbohydrates or other contaminants that absorb at or near 260, 230 or 280 nm. The miRNA content was quantitated using a Qubit microRNA assay kit (Thermo Fisher Scientific) with a Qubit 3.0 Fluorometer, according to manufacturer’s recommendations. The integrity of the RNA was determined for all samples using an Agilent Bioanalyzer 2100 (Agilent Technologies, Diegem, Belgium) with an Agilent RNA 6000 kit (Agilent Technologies). RNA Integrity Numbers (RINs) were used to evaluate the integrity of the RNA samples with > 7.0 considered intact and < 7.0 considered degraded. The quality assessment of RNA samples is provided in Additional file 1: Figure S2, Additional file 2: Figure S3, Additional file 3: Figure S4.

HMW gDNA from AUB M. zaraptor was diluted to ~0.8 ng/uL with EB buffer through a series of dilutions, with concentrations determined by Qubit 3.0 Fluorometer (Thermo Fisher Scientific, United States). The Chromium Genome Reagent Kit v2 (10× Genomics Inc., CA, United States) was used for linked-read library preparation, according to the manufacturer’s instructions. The genome chip was loaded with diluted denatured gDNA, sample master mix, and gel beads following the protocol. Gel Bead-In-EMulsions (GEMs) were generated using a 10× Chromium Controller. After the incubation and cleanup of the obtained GEMs, Chromium i7 Sample Index was ligated and served as the library barcode to provide linked information. The size distribution of the prepared library was assessed using Agilent TapeStation 4200 (Agilent Technologies, CA, United States), final library quantity was checked with Qubit 3.0 Fluorometer (Thermo Fisher Scientific, United States). After quality control, the 10× Genomic library was sequenced on an Illumina NovaSeq 6000 machine.