Gel extraction kit

Sandwich gel was scanned on Typhoon imager (Typhoon Trio and Typhoon 9410) for the detection of bands having Cy5-labelled primers applying a laser excitation at 633 nm and an emission filter at 670 nm (S2 Fig.). Then, the gel was silver stained in a 40 X 30 cm plastic tray on a shaker as described by Creste, et al. [42 ] with modifications. Silver stained and visualized differentially expressed TDFs were excised from the gel with a surgical blade and eluted in 100 μl of sterile double distilled water. They were heated at 95°C for 15 min and then, hydrated overnight at 4°C according to Baisakh, et al. [29 ]. Five μl of eluted DNA was used for re-amplification by using same primer combination and identical PCR conditions. The PCR products were examined on a 1.2% agarose gel and visualized on an UV transilluminator using ethidium bromide stain. The products were further purified using gel extraction kit (Sigma-Aldrich, USA).

A dual gRNA construct pp38-gNC, which expresses two gRNAs targeting both ends of pp38 gene and Cas9 nuclease in pX330A-1 × 2 vector was used for pp38 deletion in MSB-1 [39 (link)]. Two-part guide RNA system containing crRNA:tracrRNA guide complex was used for HP8 editing. The same gRNA sequences for pp38-gN and pp38-gC were used for synthetic crRNAs production by Integrated DNA Technologies (IDT, Diego, CA, USA). The 36-mer crRNA contains a variable gene-specific 20-nt target sequence followed by 16-nt sequence that base-pairs with the tracrRNA. The 67-mer tracrRNA contains the gRNA-scaffold sequence as well as the 16-nt sequence complementary to crRNA. The lyophilized crRNA and tracrRNA pellets were resuspended in Duplex buffer (IDT) at 200 μM concentration and stored in small aliquots at −80 °C. GFP expression cassette used for insertion into pp38 was released by PacI restriction digestion from pGEM-sgA-GFP [42 (link)] and purified by gel extraction kit (Sigma) after separation with agarose gel.

The PCR amplified gene products were extracted from gels using Gel Extraction kit (Sigma-Aldrich, St Louis, MO, USA) and the extracted DNA was quantified by nanodrop. Sequencing of genes from each isolate was performed on an ABI Prism 377 DNA Sequencer equipped with semi adaptive version 3.0. Nucleotide sequences were analysed using blast and Bio Edit Sequence Alignment Editor and compared with reference sequences of Gen-Bank accession numbers, AF314649 and AY571984 for pvcrt-o and pvmdr-1, respectively. Amplification and sequencing were repeated to confirm that the observed SNP variants were not due to PCR or sequencing errors.

Morphological and biochemical identification tests of the ZTB were carried out by using the standard protocol outlined in Bergey’s Manual of Systemic Bacteriology. Molecular identification of the ZTB was done using 16S rDNA amplification and sequencing. The universal primers for 16S r DNA viz. 27 F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1,492 R (3′-TACGGYTACCTTGTTACGACTT-5′) were used for amplification. The amplified PCR product was further purified gel extraction kit (Sigma) and sequenced directly in an automated DNA Sequencer (ABI Prism 310 Genetic Analyzer, Applied Biosystems, Inc., Foster City, CA). This was followed by assembling the sequences by BioEdit software package^{43 (link)}. Phylogenetic analysis was performed using the obtained aligned sequence followed by the BLAST with the 16S ribosomal RNA sequence (Bacteria and Archaea) nucleotide database. The closest species related to the sequence were retrieved and analyzed by MEGA software 6.0^{44 (link)}. The neighbor joining method was employed with bootstrap values generated from 1,000 replicates for construction of phylogenetic tree.

Following perfusion, mouse lung tissue was collected and submerged in RNAlater (Ambion) for 24 hours prior to long term storage at -80°C. RNA was prepared from mouse lungs using Trisure (Bioline) according to the manufacturer’s instructions (Bioline). Total RNA (2 μg) was reverse transcribed using the Tetro cDNA synthesis Kit with random primers according to the manufacturer’s instructions (Bioline). Data are expressed as fold increases over uninfected controls and were calculated by the ΔΔCT method using 18S as the reference gene.
For absolute viral nucleoprotein quantification, RNA was extracted from 1 x 10⁷ PFU PR8 using the ISOLATEII RNA kit according to the manufacturer’s instructions (Bioline) and 100 ng reverse-transcribed with the Tetro cDNA synthesis Kit using IAV nucleoprotein specific primers [39 (link)]. Following amplification, the 216 bp cDNA product was gel purified using a Gel Extraction kit (Sigma Aldrich) and total copy number determined based on size and yield of product. A standard curve was generated to determine absolute viral nucleoprotein mRNA copy number among sample mRNA.
All quantitative reverse-transcriptase PCR (qRT-PCR) was performed using SYBR NoROX master mix (Bioline) on a Roche LightCycler480. Forward and reverse qRT-PCR primers are listed in Table 1.