Qubit instrument

Total RNA was extracted from a total of 72 larvae, six per time point, per treatment. RNA was purified with the Direct-zol RNA MiniPrep (Zymo, Irvine, CA, United States) as per manufacturer’s instructions. Quality and quantity of the total RNA purified were determined using Experion equipment (Bio-Rad, Hercules, CA, United States) and a Qubit instrument (Thermo Fisher Scientific, Waltham, MA, United States) Due to technical error one individual of the PBS treatment failed, therefore this sampling point only has five replicates, which resulted in the final total of 71 individuals sequenced. Library preparation, sequencing and data processing of the RNA was performed at the National Genomics Infrastructure Sweden (NGI Stockholm) using strand specific Illumina TruSeq RNA libraries with poly-A selection (Illumina HiSeq HO mode v4, paired-end 2 × 125 bp).

Approximately 50 pM of pooled barcoded libraries were used for templating using Thermo Fisher Ion 550 Kit-Chef (ThermoFisher, MA, United States, Cat.# A34541) and the manufacturer’s recommended protocol. Briefly, 50 pM of pooled libraries were combined and 25 ul of each sample was loaded onto the Ion Chef. Next, all reagents for the Ion 550 Chef Kit were loaded onto the Ion Chef (ThermoFisher, MA, United States) and the run was performed. The Ion Chef templated, enriched, and loaded the sample onto a 550 chip. After 15 h the Chef paused, and QC was performed on the unenriched samples. The beads were then isolated, and quality assessment was performed on the Qubit instrument (ThermoFisher, MA, United States) to determine the percentage of beads that were polyclonal. All samples at this point passed QC. After polyclonal assessment the Ion Chef resumed running and loaded the samples onto a 550 chip. The loaded chip was then placed into an Ion S5 sequencer, and the run was started using an Ion torrent Ampliseq transcriptome run plan that was configured based on type of library, species, number of run flows required, type of plug-in required, adapter-trimming as well as other parameters specific to the Ampliseq transcriptome run.

Biopsy specimens were processed and RNA purified as previously described [11 (link)].
RNA-seq was carried out at the National Genomics Infrastructure node in Stockholm (Sweden). The concentration of the RNA samples were measured using a Qubit instrument and the Qubit RNA BR assay (Thermofisher Scientific, Waltham, MA, USA), and Agilent RNA Nano chip was used to assess the integrity of the samples (Agilent Technologies, Santa Clara, CA, USA). Libraries for sequencing were constructed using the TruSeq stranded total RNA kit with Ribo-Zero for depletion of ribosomal RNA (Illumina Inc., San Diego, CA, USA). Clustering was accomplished using the cBot system and samples were sequenced on HiSeq2500 with a 1 × 51 nucleotides setup using the HiSeq SBS Kit v4 chemistry (Illumina). Sequencing data were converted to the fastq format and demultiplexed using the bcl2fastq2 conversion software (Illumina Inc., San Diego, CA, USA).

Total RNA and DNA was extracted from five archived filters for metatranscriptomics and amplicon sequencing analysis collected after 24 h acclimation (T0), 4 days (T4C and T4O) and 7 days (T7C and T7O). The extraction was carried out using the AllPrep Bacterial DNA/RNA/Protein Mini Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. All samples were eluted in 50 µL volume. RNA concentration was quantified on a Qubit instrument (Thermo Fisher Scientific, Carlsbad, CA, USA) and the quality of the samples was evaluated on agarose gel.

Upon return to the laboratory samples were concentrated by vacuum filtration first through a 5.0 μm nitrocellulose (Millipore) then 0.2 μm nitrocellulose filter (Millipore) then stored at −80°C until DNA extraction. Total genomic DNA from each 0.2 μm filter was extracted using the MoBio PowerWater kit according to the manufacturer’s instructions. Nucleic acid quality (i.e., 260/280 ratio) was measured with a Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, United States). DNA concentrations for all samples were measured by fluorometric quantitation using a Qubit^® instrument and High Sensitivity dsDNA HS Assay kit (Thermo Fisher Scientific, Waltham, MA, United States); and purified DNA extracts were stored at −80°C until used.