Qubit 3.0 fluorimeter

Heart samples were homogenized in a TissueLysser LT (Qiagen). RNA extractions were performed using a Quik-RNA^TM miniprep plus kit (Zymo Research) for non-coding RNA sequencing (ncRNA-seq; ICM, n = 22; control, n = 8) or a PureLink™ Kit (Ambion Life Technologies) for mRNA sequencing (mRNA-seq; ICM, n = 13; control, n = 10), according to the manufacturer’s instructions. RNA was quantified using a NanoDrop1000 spectrophotometer and at Qubit 3.0 fluorimeter (Thermo Fisher Scientific). The purity and integrity of RNA samples was measured using an Agilent 2100 Bioanalyzer with the RNA 6000 Nano LabChip kit (Agilent Technologies). All samples displayed a 260/280 absorbance ratio > 2.0 and RNA integrity numbers ≥ 9.

Sequencing was performed on Illumina NextSeq 550. For a single sequencing run, a 4-multiplexed library was created by pooling the libraries, quantified using QuBit dsDNA HS Assay kit with QuBit 3.0 fluorimeter (Thermo Fisher Scientific, San Jose, CA) and qPCR with QIAseq Library Quant Assay Kit (QIAgen, Hilden, Germany), at an equal molar ratio. The multiplexed library was denatured and sequenced with NextSeq High Output kit 2 ×150 cycles paired.

Total DNA was isolated from 1 mL of bacterial culture using the Genomic Mini Kit (A&A Biotechnology) according to the manufacturer’s protocol. The amount of DNA was determined using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific) on a Qubit 3.0 fluorimeter (Thermo Fisher Scientific) according to the manufacturer’s protocol.

RNA was extracted from 500-mg rind sample aliquots without prior separation of microbial cells, as previously described (Monnet et al., 2012 (link)), except that the DNase treatment was performed on the RNeasy spin columns (Qiagen, Courtaboeuf, France). Purified RNA was quantified with Qubit RNA assay kits on the Qubit 3.0 fluorimeter (ThermoFischer Scientific). RNA quality was analyzed with an Agilent model 2100 Bioanalyzer (Palo Alto, CA, USA) using RNA 6000 NANO chips, according to the manufacturer's instructions. For each sample, 10 μg of total RNA was then subjected to rRNA depletion using the Epicentre Ribo-Zero™ Magnetic Gold Kit (Tebu-bio, Le Perray-en-Yvelines, France) for bacteria (reference MRZB12424) and for yeasts (reference MRZY1324). Depletion was performed according to the manufacturer's instructions, except that a mixture (50/50) of the yeast and bacteria Ribo-Zero rRNA solutions was used. The rRNA-depleted samples were then purified using a RNeasy MinElute Cleanup Kit (Qiagen), according to the modified procedure described in the Ribo-Zero™ Magnetic Gold Kit technical procedure. The quality of depleted RNA was assessed on an Agilent model 2100 Bioanalyzer, using RNA 6000 PICO chips.

We cut two consecutive sections (4 μm and 8 μm thick, respectively) from each FFPE tissue block in the discovery and validation cohorts and used the 4-μm-thick section for hematoxylin and eosin staining and the 8-μm-thick section for gDNA extraction. To extract gDNA, we used the QIAamp DNA FFPE Tissue Kit (Qiagen, Germany) following the manufacturer’s protocol. We assessed the quality and quantity of the gDNA samples using a NanoDrop 2000 and a Qubit 3.0 fluorimeter (Thermo Fisher Scientific, Germany) and retained only samples that had a range of light absorption (A₂₆₀/A₂₈₀) comprised between 1.8 and 2.0. We sheared 6 to 10 ng of each purified gDNA using a Bioraptor Plus (Diagenode, Germany) with cycling conditions optimized to achieve a mean target size of 150 to 200 base pairs (bp). We evaluated the distribution of the sheared gDNA on a Bioanalyzer 2100 (Agilent Technologies, UK) using a High Sensitivity DNA Kit. We remeasured the quantity of the sheared gDNA on a Qubit fluorometer and stored the samples at −20°C until we prepared sequencing libraries.

All positive samples were submitted to a cDNA synthesis protocol using Protoscript II First Strand Sequencing kit (New England Biolabs—NEB). Then, a multiplex PCR was conducted using Q5 High Fidelity Hot-Start DNA Polymerase (New England Biolabs) and a sequencing primer scheme (divided into two separated pools) designed using Primal Scheme online tool to amplify 400 bp overlapping amplicons of the CHIKV complete genome (http://primal.zibraproject.org) [12 (link)]. All samples were subjected to 45 cycles of PCR using the thermocycling conditions of [12 (link)]. PCR products were purified using a 1x SPRI bead cleanup (Ampure XP Beads Agencourt) and concentrations were measured using a Qubit dsDNA High Sensitivity kit on a Qubit 3.0 fluorimeter (ThermoFisher).

DNA was quantified using a Qubit 3.0 Fluorimeter (Thermo Fisher Scientific, MA, USA) following the manufacturer’s protocol, and was found to be 12.7 ng·ml⁻¹. 7.5 µl of this DNA was used for library preparation using the RAD002 kit (Oxford Nanopore Technologies, Oxford, UK). Since the input quantity of DNA was lower than recommended for this kit, the active FRM reagent was diluted with three volumes of heat-inactivated FRM, to avoid over-fragmentation of the DNA. The library preparation reaction was set up as follows: the reaction (DNA 7.5 µl, 0.25 × FRM 2.5 µl) was incubated for 1 min at 30 °C followed by 1 min at 75 °C. We added 1 ml of RAD reagent from the RAD002 kit and 0.2 ml of Blunt TA ligase (New England Biolabs, MA, USA) and the reaction was incubated for 5 min at room temperature. The prepared library was then loaded onto a FLO-MIN106 flowcell (version 9.4 nanopores) as per Oxford Nanopore Technologies’ standard protocol.