Transcriptome Sequencing of Virus Infection Protocol

Infection of Cells

1. Seed CRFK cells into 75 cm² flasks and incubate at 37 °C with 5 % CO₂ until cells reach 60–70 % confluency.
2. Remove media and wash the cells with D-PBS.
3. Infect the flasks with virus at multiplicity of infection (MOI) 2 in 2 ml, or 2 ml D-PBS as a mock control, and incubate at 37 °C with 5 % CO₂ for 1 h to allow attachment. Perform inoculations in duplicates, one for RNA extraction and the other for CPE visualization.
4. Add 10 ml of maintenance medium with 10 % FBS and incubate the flasks for 3 h.
5. Following 3 h of incubation, remove inoculum, wash the cells with D-PBS.
6. Add 2 ml TrypLE and incubate for 1–2 min until cells detach.
7. Transfer cells to a centrifuge tube and pellet the cells by centrifugation at 120 × g for 5 min and discard supernatant.
8. Add 10 ml D-PBS and repeat centrifugation in order to remove every trace of medium and TrypLE, which could reduce RNA yield.
9. Discard the supernatants and store the cell pellets at −80 °C until RNA purification.

RNA Purification from Infected Cells

The RNeasy kit was used to extract and purify RNA samples in this study but other RNA extraction protocols may also be suitable.
1. Spray all micropipettes, gloves, working area, and other things with RNase AWAY to remove any RNase and DNA contamination.
2. Extract RNA using RNeasy kit according to the manufacturer's instruction.
3. Aliquot the eluted RNAs (500 μl) into three different tubes to avoid repeated thawing and freezing of the sample which could affect the quality of the RNA.
4. Use two tubes for quality control analysis with spectrophotometer and Illumina Agilent 2100 bio-analyzer and store the third one at −80 °C for sequencing.

Total RNA Quality Analysis

1. Determine the quality of extracted RNA by measuring the absorbance at 260 and 280 nm in UV/Visible spectrophotometer.
2. Consider the samples with the absorbance ratio value (A260/A280) of 1.8 to 2.0 for further analysis with Illumina Agilent 2100 Bio-analyzer to determine both the RNA quality and quantity.
3. In order to ensure the samples are of highest quality and quantity for transcriptome sequencing, conduct quality and quantity analysis to the extracted total RNA samples.
4. Load and prime gel–dye mixtures, then load marker, ladder, and samples in the specified manner.
5. Vortex the chip and insert. Analyze the chips based on the method recommended by the manufacturer. Verify whether the run is successful and whether the sample is properly prepared and handled by means of properly pipetted into the wells.

Preparing Samples for Paired-End Sequencing

Perform the following steps using the reagents provided in the paired end sample preparation kit, according to manufacturer's instructions.
1. Fragment genomic DNA into fragments of less than 800 bp.
2. Perform end repair of DNA fragments to generate 5'-phosphorylated blunt ends.
3. Add an "A" base to the 3' ends to make 3'-dA overhang.
4. Ligate adapters to the ends of the DNA fragments.
5. Purify ligation products by removing un-ligated adapters.
6. Enrich the Adapter-Modified DNA Fragments by PCR.
7. Obtain the Genomic DNA library.