Qubit 2.0 flourometer

For 107 patients, we performed WES on tumor DNA and constitutive normal DNA. DNA was extracted using the QIAamp DNA kit (Qiagen) according to manufacturer’s protocol. DNA quantification was performed on a Qubit 2.0 Flourometer (Life Technologies). Libraries for WES were prepared on the SureSelect Automated Library Prep and Capture System (Agilent Technologies) according to the manufacturer’s protocol (version E.3). In brief, genomic DNA (1.5–3 μg) from each sample was fragmented to a length distribution peak of 150 to 200 nt for the preparation of paired-end sequencing libraries. Enrichment for exomic sequence was performed using Agilent SureSelect V4+UTR in-solution capture reagents following vendor’s protocol v2.0.1. Sequencing was carried out on HiSeq 2000 machines (Illumina) with 3 samples multiplexed per lane.
For RNA-Seq, RNA was extracted from 123 patients using the RNA RNeasy mini kit (Qiagen) according to the manufacturer’s protocol. RNA quantification was performed on a Qubit 2.0 Flourometer. Quality was assessed on an Agilent 2100 Bioanalyzer. An RNA integrity number (RIN) of at least 8 was required. RNA-Seq libraries were prepared according to the manufacturer’s protocol (Illumina TruSeq RNA sample preparation v2). Sequencing was performed on Illumina HiSeq 2000 machines with 2–3 samples multiplexed per lane.

Swabs were resuspended in 200 µL extraction buffer (20 mM Tris-HCl ph7.5, 150 mM NaCl, 1 mM PMSF, 0.05% Tween 20 and 1 uL/mL protease inhibitor cocktail (Roche), vortexed, incubated overnight at 4°C, swab removed and then 50 µL more extraction buffer was added before being stored at −80°C. The resulting samples were thawed on ice and loaded onto a Milliplex Human High Sensitivity Cytokine/Chemokine Panel (EMD Millipore, Billerica, Mass, USA) and analyzed for IL-1B, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p70, IL-13, IFNg, GM-CSF, and TNFa. Results for IL-2, IL-7, IL-8, IL-10, IL-13, IFNg were not displayed as they were consistently outside the assay range and there was limited material for reanalysis. The plate was analyzed using a Bio-Plex 200 System (Bio-Rad Laboratories, CA, USA) with cytokine/chemokine levels being generated automatically from standard curves using the Bio-Plex Manager software (v.4.1.1 Bio-Rad). In cases where the analyte was detected, but below the limit of detection, ½ the LOD was used for analysis. Results were normalized to total protein as determined with the Qubit Protein Assay Kit and Qubit 2.0 Flourometer (Life Technologies, CA, USA). Paired t-tests were used to make comparisons.

NLHS and bacterial challenge experiments were performed as described above and approximately 500 µL of hemolymph of VP_AHPND-challenged NLHS-treated and VP_AHPND-challenged NH control shrimp at 0, 6, and 24 h post infection (hpi) time points were drawn out from the ventral sinus using a sterile syringe pre-loaded with an equal volume of anticoagulant (27 mM sodium citrate, 336 mM NaCl, 115 mM glucose, and 9 mM EDTA, pH 5.6)^{37 (link)}. Hemocytes were immediately collected by centrifugation at 800 × g for 10 min at 4 °C and kept in liquid nitrogen. The hemocytes from 30 individuals were pooled and extracted for large and small RNAs using the mirVana miRNA Isolation Kit (Ambion, Life Technologies) following the manufacturer’s protocol. These experiments were done using triplicates. RNA integrity was evaluated using the Agilent 2100 Bioanalyzer chip, RNA 6000 Pico Kit and Small RNA Kit (Agilent) for large and small RNA preparations, respectively. The RNA concentrations were determined using the Qubit RNA HS Assay Kit on the Qubit 2.0 flourometer (ThermoFisher Scientific).