Agilent 2100 bioanalyser system

RNAzol isolation method allows for extraction of RNA without the use of a chloroform phase separation step. The RNAzol protocol for recovery of total RNA was adapted and optimized as follows: a volume of 400 µL of sputum collected from SS_whole, SS_MTM_plug or SS_MTM_whole was mixed with 1 ml of RNAzol and incubated for 15 min at room temperature. Following centrifugation at 16,000 × g for 15 min, the DNA, proteins and most polysaccharides were pelleted at the bottom of the tube. The supernatant was recovered, and 1 ml volume of isopropanol was added to precipitate the RNA. Following a series of ethanol wash steps, the crude RNA was quantified using nanodrop™ (Thermo Fisher Scientific, Massachusetts, United States) and purified through incubation with DNaseI followed by filter purification using the RNeasy® MinElute® spin column (Qiagen, Hilden, DE) to yield total RNA in a volume of 14 µL (molecular grade water). Samples were extracted in triplicate to maximise the RNA yield. The extracted RNA samples were then assessed for concentration and purity using the Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific, Massachusetts, United States). Quality was assessed using the Agilent Bioanalyser 2100 system (Agilent, Santa Clara, United States). Fluorescent dye-based quantification of RNA (Qubit, Life Technologies) was utilized to confirm the presence of RNA.

Amplicon sequencing was done at the Novogene company, China, following the standard company protocol. Precisely, after diluting the DNA to 1 ng/μL in sterile water, fungal ITS genes of distinct regions (ITS2) were amplified using a specific set of primers (ITS3, ITS4; amplicon size 386 bp) (White et al., 1990 (link)) with unique barcodes. PCR reactions were performed using Phusion High-Fidelity PCR Master Mix (New England Biolabs). PCR products with amplification between 400 and 450 bp were selected and mixed in equidensity ratios for gel purification. No template control in PCR reaction did not show any amplification; hence it was not used for gel purification and further library preparation. After purification of PCR products using the Qiagen Gel Extraction Kit (Qiagen, Germany), sequencing libraries were created using NEBNext Ultra DNA Library Pre-Kit from Illumina, and index codes were ligated. The library quantity and quality were analyzed using Qubit 2.0 Fluorometer (Thermo Fisher Scientific), Q-PCR and in Agilent Bioanalyser 2100 system respectively. The libraries were sequenced to generate 250 bp paired-end reads using an Illumina platform.

Leaf samples collected at four stages (i.e., PRE, ES, IS and LS) except ABS were submitted for RNA-Seq, because no high-quality RNA could be extracted from samples at ABS stage. Total RNA samples were extracted using the TRIzol reagent (Invitrogen) and then treated with RNase-free DNase I (Takara, Beijing, China) to remove genomic DNA. The quantity and quality of RNAs were tested using a Nanodrop 2000 spectrophotometer (NanoDrop Tchnologies, Wilmington, ED, USA) and Agilent Bioanalyser 2100 system (Agilent Technologies, Palo Alto, CA, USA), respectively. The mRNAs were first purified from RNA samples using poly-T oligo-attached magnetic beads. Strand-specific RNA-Seq library preparation and sequencing were both performed by Novogene Biotech (Beijing, China) using standard Illumina protocols on an Illumina HiSeq 2500 system with the paired-end mode. Adaptor sequences and low-quality sequences were removed from the raw reads (Q < 20). Clean reads were assembled using the Cufflinks v2.1.1 reference annotation-based transcript (RABT) assembly method [55 (link)] and mapped to the reference genome of Populus trichocarpa (https://phytozome.jgi.doe.gov/pz/portal.html accessed on 17 August 2019) to get gene annotations using HISAT (2.1.0) software [56 (link)].

Total RNA was extracted from the breast tissue using miRNeasy Mini Kit (Qiagen, Germany) as recommended by the manufacturer. RNA concentrations were determined by the Nanodrop photometer (NanoDrop, USA). RNA quality was checked using the Agilent Bioanalyser 2100 System (Agilent Technologies, USA). For all samples RNA integrity number (RIN) was greater than 7.

Three pools—each containing whole organs from approximately 20 mosquitos—were used for total RNA extraction using Trizol reagent (Invitrogen, San Diego, CA, USA). For infected organs, we extracted the mosquito and parasite RNA simultaneously. RNA was amplified and labeled using two rounds of linear amplification according to the manufacturer's protocol (GeneChip® two-cycle cDNA synthesis kit; Affymetrix, Santa Clara, CA, USA). RNA quality and purity before and after amplification were assessed by high-resolution electrophoresis using the Agilent 2100 Bioanalyser system (Agilent Technologies, Inc., Santa Clara, CA, USA).

All experiments were performed in three replicates with a total number of 600 larvae per replicate. Total RNA extraction from microglial cells was performed using the Qiagen RNeasy Plus Micro kit according to the manufacturer's guidance (Qiagen). RNA sample quality and concentration were determined using Agilent RNA 6000 Pico kit and an Agilent 2100 Bioanalyser System (Agilent Technologies).
For sequencing, all RNA samples with a RIN score >7 were transcribed into cDNA using the Ovation RNA‐Seq System V2 kit according to the manufacturer's instructions (NuGEN). Samples were then sent to Edinburgh Genomics for library synthesis and sequencing. For qPCR, RNA sample quality and concentration were assessed using the LabChip GX Touch Nucleic Acid Analyzer and RNA Pico Sensitivity Assay. All RNA samples with a RIN score >7 were transcribed from the same amount of RNA into cDNA using the Super‐Script® III First‐Strand Synthesis System (Invitrogen).