Qubit fluorometer

DNA concentrations were measured using the Qubit Fluorometer and the Qubit dsDNA BR Assay Kit (ThermoFisher Scientific, Loughborough, UK) and 250 ng were used for sequencing library preparation. Indexed sequencing libraries were prepared using the Nextera DNA Flex Library Prep Kit (Illumina, San Diego, CA, USA) and Nextera DNA CD Indexes (Illumina, San Diego, CA, USA). Libraries were quantified using the Qubit Fluorometer and the Qubit dsDNA HS Kit (ThermoFisher Scientific, Loughborough, UK). Library fragment-length distributions were assessed using the Agilent TapeStation 4200 and the Agilent High Sensitivity D1000 ScreenTape Assay (Agilent, Santa Clara, CA, USA). Libraries were pooled in equimolar amounts and final library quantification performed using the KAPA Library Quantification Kit for Illumina (Roche, Basel, Switzerland). The library pool was sequenced on an Illumina MiSeq using the MiSeq Reagent Kit v2 (500 cycle) (Illumina, San Diego, CA, USA) to generate 250 bp paired-end reads. The whole genome shotgun was deposited in GenBank as a BioProject under Accession PRJNA826427.

Briefly, cfDNAs were isolated from 2.0 ml to 9.6 ml of plasma using MagMAX Cell-Free Total Nucleic Acid Isolation kit (Thermo Fisher Scientific, Waltham, MA) for panel assay or using MagMAX Cell-Free DNA Isolation kit (Thermo Fisher Scientific) for ddPCR assay, according to the manufacturer’s instruction. The cfDNA concentration was quantified using Qubit fluorometer (Thermo Fisher Scientific), while its quality was examined by Bioanalyzer or Tapestation system (Agilent Technologies, Santa Clara, CA).
For the detection of CH-derived mutation, gDNA was isolated from the buffy coat fraction using QIAamp DNA Blood Mini Kit (Qiagen, Germantown, MD). Both quantity and quality of gDNA were examined by NanoDrop (Thermo Fisher Scientific) as well as by Qubit fluorometer (Thermo Fisher Scientific) and Tapestation (Agilent Technologies).

Total DNA was extracted in the robotic workstation MagNA Pure LC Instrument (Roche) using the MagNA Pure LC DNA isolation kit III (Bacteria, Fungi) (Roche). Previously, salivary samples (300 μL of saliva) were centrifuged at maximum speed at 4 °C for 5 min to pellet the cells. Bacterial pellets were resuspended in lysis buffer (MagNA Pure LC DNA isolation kit III) and treated for one hour at 37 °C with an enzymatic cocktail (lysozyme 25 mg/mL, lysostaphin 1.25 mg/mL, mutanolysin 625 U/mL). Then, the samples were treated for 15 min at 65 °C with proteinase K according to the instructions of MagNA Pure LC DNA isolation kit III. Total DNA was quantified with a Qubit Fluorometer (ThermoFisher) and its integrity was verified by standard agarose gel electrophoresis.
We amplified the V3–V4 region of the 16S rRNA gene from total DNA obtained from salivary and fecal samples. The amplicon libraries were constructed following Illumina instructions. The libraries were quantified with a Qubit Fluorometer (ThermoFisher) and sequenced using the Kit v3 (2 × 230 cycles) in a MiSeq platform (Illumina) at FISABIO-Salud Pública. All the sequences were deposited in the European Bioinformatics Institute (EBI) database under the number PRJEB25569.

Flasks containing a final concentration of 10⁵ blastospores/mL in YSB or C-depleted medium were incubated for four days at 18 °C to induce blastosporulation or mycelial growth, respectively. Total RNA was extracted from three replicates per strain and condition by using TRIzol (Thermo Fisher Scientific, Waltham, Massachusetts) following the manufacturer’s instructions. RNA concentration and quality were checked using a Qubit fluorometer (Thermo Fisher Scientific, Waltham, Massachusetts) and a TapeStation 2200 (Agilent, Santa Clara, California). Only samples showing good ribosomal RNA bands without RNA degradation and no DNA contamination were used for sequencing. A Truseq stranded mRNA kit (Illumina Inc., San Diego, CA, USA) was used for library preparation, and ribosomal RNA was depleted by polyA enrichment. The quality and quantity of the enriched libraries were assessed using a Qubit fluorometer (Thermo Fisher Scientific) and TapeStation (Agilent). Libraries were sequenced on the Illumina HiSeq 2500 using 2 × 100 bp paired-end reads.

Total RNA was isolated from roots, leaves, flowers, developing seeds, and pneumatophores of A. marina using RNeasy Plant Mini Kit (Qiagen, Germany). The RNA was purified after DNase I treatment using the RNeasy MinElute Cleanup Kit (Qiagen, Germany). The quality and quantity of the RNA were assessed using a spectrophotometer, Qubit Fluorometer (ThermoFisher, USA), and 2100 Bioanalyzer (Agilent, USA). SMARTer PCR cDNA Synthesis Kit was used, and cDNA was prepared as per the manufacturer’s protocol (Takara, USA). SMRTbell library was prepared using SMRTbell Template Prep Kit 1.0 (Pacific Biosciences, CA, USA), and size-selected using the BluePippin size-selection system (Sage Science, MA, USA). The size-selected library was purified and assessed using Qubit Fluorometer (Invitrogen, USA) and FEMTO Pulse System (Agilent, USA). The DNA/Polymerase Binding Kit P6 was used for the binding of the DNA polymerase P6 to the cDNA templates in the SMRTbell library. The polymerase-bound SMRTbell library was subjected to single-molecule real-time (SMRT) sequencing using SMRT cells (SMRT 1M v3 LR) and Sequel DNA Sequencing Kit 3.0 with P6-C4 chemistry in PacBio Sequel System (Pacific Biosciences, CA, USA).

Total RNA was isolated from roots, leaves, flowers, developing seeds, and pneumatophores of A. marina using RNeasy Plant Mini Kit. The RNA was purified after DNase I treatment using the RNeasy MinElute Cleanup Kit (Qiagen, Germany). The quality and quantity of the RNA were assessed using a spectrophotometer, Qubit Fluorometer (ThermoFisher, USA), and 2100 Bioanalyzer (Agilent, USA). Separate RNA-Seq libraries were prepared for the RNA from different tissues using NEBNext Ultra II RNA Library Prep Kit (NEB, USA). Messenger RNA was purified from total RNA using Oligo-dT magnetic beads and fragmented with fragmentation buffer. Random hexamers were used to synthesize double-strand cDNAs from the fragmented mRNAs. The cDNAs were blunt-ended by end-repair, and sequencing adapters were ligated. The library was enriched by amplification using the sequencing primers and assessed for its insert size using 2100 Bioanalyzer (Agilent, USA). The library was quantified using Qubit Fluorometer (Invitrogen, USA), diluted to 4.0 nM concentration, and denatured using sodium hydroxide. The libraries were sequenced with paired-end (2 × 150 bp) sequencing chemistry using the NextSeq 500 platform (Illumina, USA).