Lysing matrix e 2 ml tubes

Three biological replicate experiments were used for RNA extraction and analyses at each time point. RNA was extracted with MO BIO PowerWater RNA Isolation Kit [MO BIO Laboratories (now QIAGEN), Carlsbad, CA, United States] following a 2-min bead beating step using Lysing Matrix E 2 mL tubes (MP Biomedicals, Santa Ana, CA, United States). The manufacturer’s protocol was followed with slight modification: specifically, the DNAse treatment step was performed twice to ensure sufficient purity of the RNA. RNA was quantified using a Nanodrop ND-1000 Spectrophotometer (Thermo Scientific, Waltham, MA, United States) and RNA integrity was checked with an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, United States). Extracted RNA was processed and sequenced at the Hudson Alpha Genomic Services Lab (Huntsville, AL, United States). RNA samples were poly-A selected to enrich for mRNA and deplete ribosomal RNA transcripts. Samples were sequenced using an Illumina^® NextSeq^® sequencer targeting approximately 25 million single-end reads per sample and a 76-bp read length. Standard protocols by Illumina^® were followed for library preparation, poly-dT bead selection, and sequencing. Sequence data have been deposited in the NCBI short-read archive under bioproject PRJNA432024.

Tissue samples were weighed (≈20 mg) and placed into Lysing Matrix E 2 mL tubes (MP Biomedicals™, Santa Ana, CA, USA) for disruption and homogenization with RLT-Plus buffer/β-mercaptoethanol (600 μL) in a TissueLyser II (Qiagen, Venlo, Netherlands) for 2 min. The lysate was centrifuged at full speed, and the supernatant was used to extract total RNA using the RNeasy Plus Kit (Qiagen) as per the manufacturer’s instructions. Total RNA was eluted in a final volume of 40 µL of RNase-free water. From the RNA extracts, 24 samples were sent for the sequencing of messenger RNA (mRNA). These 24 samples were selected among the four experimental groups described in Section 4.6 (controls, POMV-positives, POMV-suspects, and survivors), randomly sampling three fish per group and collecting two different tissues (liver and head kidney) from each fish. Sequencing reads were produced as 100-bp paired-end reads across three lanes on a HiSeq 2000 sequencer (Illumina, San Diego, CA, USA) by Macrogen (Seoul, Korea) using the TruSeq stranded mRNA protocol.

Female 8- to 10-week-old Ifngr1^-/- mice from Jackson Laboratories were orally gavaged with 200 μl of bleached ALI material (equivalent to 4-5 transwells) from either day 1 (n = 4 mice) or day 3 (n = 4 mice) post-infection cultures. After gavaging, mice were housed separately for the duration of the experiment to avoid cross-infection. One mouse infected with bleached, day 1 ALI culture material was sacrificed on day 30 post-infection and one mouse infected with bleached, day 3 ALI culture material was sacrificed on day 9 post-infection to collect the small intestine for histology as described above. Mouse pellets were collected every 2-3 days, and the mice were monitored for signs of sickness. Mouse pellets were kept at -80°C until they were processed for DNA extraction, which was performed using the QIAamp DNA Stool Kit (QIAGEN). Pellets were moved to Lysing Matrix E 2 ml tubes (MP Biomedicals) and 1.4 ml ASL Buffer (from kit) was added. Samples were homogenized using the FastPrep-24^™ 5G High-Speed Homogenizer, then processed according to the kit’s directions. qPCR was used to quantify the number of C. parvum genomic DNA equivalents present in the sample using the C. parvum GAPDH primers and cycling protocols as described above.