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• step description "Cells were seeded in 100-mm-diameter dishes and infected with either 2 PFU of live IBV per cell or the same amount of UV-inactivated IBV (UV-IBV) at 37 °C. Excess virus in the medium was removed by replacing with fresh medium at 1 h post-infection." assertion.
• step description "Cells were seeded in 100-mm-diameter dishes and infected with either 2 PFU of live IBV per cell or the same amount of UV-inactivated IBV (UV-IBV) at 37 °C. Excess virus in the medium was removed by replacing with fresh medium at 1 h post-infection." assertion.
• step description "At the indicated time points (0, 2, 4, 8, 12, 16, 20, 24, 28 h post-infection), cells were rinsed with 10 ml Phosphate Buffered Saline (PBS) buffer once and lysed in 1 ml TRIzol for 5 min at room temperature." assertion.
• step description "At the indicated time points (0, 2, 4, 8, 12, 16, 20, 24, 28 h post-infection), cells were rinsed with 10 ml Phosphate Buffered Saline (PBS) buffer once and lysed in 1 ml TRIzol for 5 min at room temperature." assertion.
• step description "At the indicated time points (0, 2, 4, 8, 12, 16, 20, 24, 28 h post-infection), cells were rinsed with 10 ml Phosphate Buffered Saline (PBS) buffer once and lysed in 1 ml TRIzol for 5 min at room temperature." assertion.
• step description "Cell lysates were transfer into eppendorf tubes and one-fifth (volume/volume) of chloroform was added to each tube." assertion.
• step description "Cell lysates were transfer into eppendorf tubes and one-fifth (volume/volume) of chloroform was added to each tube." assertion.
• step description "Shake tubes vigorously by hand for 15 sec and incubated for 3 min at room temperature, then centrifuged at 12,000 x g for 15 min at 4 °C. " assertion.
• step description "The upper aqueous phase was transfer into a new tube and mixed with 1:1 (volume/volume) of 100% isopropanol, and then incubated for 10 min at room temperature." assertion.
• step description "The upper aqueous phase was transfer into a new tube and mixed with 1:1 (volume/volume) of 100% isopropanol, and then incubated for 10 min at room temperature." assertion.
• step description "The upper aqueous phase was transfer into a new tube and mixed with 1:1 (volume/volume) of 100% isopropanol, and then incubated for 10 min at room temperature." assertion.
• step description "RNA was precipitated by centrifugation at 12,000 x g for 10 min at 4 °C." assertion.
• step description "RNA was precipitated by centrifugation at 12,000 x g for 10 min at 4 °C." assertion.
• step description "RNA was precipitated by centrifugation at 12,000 x g for 10 min at 4 °C." assertion.
• step description "RNA was precipitated by centrifugation at 12,000 x g for 10 min at 4 °C." assertion.
• step description "RNA pellet was washed with 1 ml 70% RNase-free ethanol once and spin down by 7,500 x g for 5 min" assertion.
• step description "The RNA pellets are air-dried and dissolved in 100 µl RNase-free H2O by incubating at 65 °C for 15 min" assertion.
• step description "RNA concentration and purity were determined by NanoDrop" assertion.
• step description "The RNAs were stored at -80 °C for further use" assertion.
• step description "After incubating at room temperature for a few minutes, keep the solubilized nucleic acid in −20˚C for a short time storage or in −80˚C for a longtime storage. Figure 1 shows a schematic model of all the DNA and RNA isolation steps of this procedure." assertion.
• step description "After incubating at room temperature for a few minutes, keep the solubilized nucleic acid in −20˚C for a short time storage or in −80˚C for a longtime storage. Figure 1 shows a schematic model of all the DNA and RNA isolation steps of this procedure." assertion.
• step description "After incubating at room temperature for a few minutes, keep the solubilized nucleic acid in −20˚C for a short time storage or in −80˚C for a longtime storage. Figure 1 shows a schematic model of all the DNA and RNA isolation steps of this procedure." assertion.
• step description "In this step, incubate samples at 65℃ for 10 min for lysing cells completely." assertion.
• step description "In this step, incubate samples at 65℃ for 10 min for lysing cells completely." assertion.
• step description "In this step, incubate samples at 65℃ for 10 min for lysing cells completely." assertion.
• step description "Transfer the resulting solution to a sterile centrifuge tube (size=2 ml), and then mix sample by briefly vortexing until the sample is thoroughly resuspended." assertion.
• step description "Transfer the resulting solution to a sterile centrifuge tube (size=2 ml), and then mix sample by briefly vortexing until the sample is thoroughly resuspended." assertion.
• step description "Transfer the resulting solution to a sterile centrifuge tube (size=2 ml), and then mix sample by briefly vortexing until the sample is thoroughly resuspended." assertion.
• step description "Transfer the resulting solution to a sterile centrifuge tube (size=2 ml), and then mix sample by briefly vortexing until the sample is thoroughly resuspended." assertion.
• step description "Ground samples (leaf, shoot, root, and recalcitrant samples, approximately 0.5-1 g) using 1 ml of the extraction buffer with or without liquid nitrogen in mortar and pestle that are sterilized.CRITICAL STEP The procedure are carried out at room temperature except the centrifugation steps (at 4℃) as well as the time of precipitating of the nucleic acid using the isopropanol (on ice).CRITICAL STEP Severely disrupt the tissues to create the glaze mode of samples." assertion.
• step description "Ground samples (leaf, shoot, root, and recalcitrant samples, approximately 0.5-1 g) using 1 ml of the extraction buffer with or without liquid nitrogen in mortar and pestle that are sterilized.CRITICAL STEP The procedure are carried out at room temperature except the centrifugation steps (at 4℃) as well as the time of precipitating of the nucleic acid using the isopropanol (on ice).CRITICAL STEP Severely disrupt the tissues to create the glaze mode of samples." assertion.
• step description "Dissolve the pellet in 20-30 µl of RNase free water (commercial) or autoclaved water." assertion.
• step description "Dissolve the pellet in 20-30 µl of RNase free water (commercial) or autoclaved water." assertion.
• step description "Dissolve the pellet in 20-30 µl of RNase free water (commercial) or autoclaved water." assertion.
• step description "Dissolve the pellet in 20-30 µl of RNase free water (commercial) or autoclaved water." assertion.
• step description "Dissolve the pellet in 20-30 µl of RNase free water (commercial) or autoclaved water." assertion.
• step description "After centrifuging at 5400 g at 4˚C for 5 min, remove the supernatant and then air-dry the pellet." assertion.
• step description "After centrifuging at 5400 g at 4˚C for 5 min, remove the supernatant and then air-dry the pellet." assertion.
• step description "After centrifuging at 5400 g at 4˚C for 5 min, remove the supernatant and then air-dry the pellet." assertion.
• step description "After centrifuging at 5400 g at 4˚C for 5 min, remove the supernatant and then air-dry the pellet." assertion.
• step description "After centrifuging at 5400 g at 4˚C for 5 min, remove the supernatant and then air-dry the pellet." assertion.
• step description "Discard the supernatant and wash the precipitate nucleic acid gently with 70% EtOH." assertion.
• step description "Discard the supernatant and wash the precipitate nucleic acid gently with 70% EtOH." assertion.
• step description "Discard the supernatant and wash the precipitate nucleic acid gently with 70% EtOH." assertion.
• step description "Centrifuge tubes at 13700 g at 4℃ for 10 min. The white pellet will be visible on the bottom of the tubes.CRITICAL STEP Do not disturb bottom phases of the solution when you pipet the upper phase." assertion.
• step description "Centrifuge tubes at 13700 g at 4℃ for 10 min. The white pellet will be visible on the bottom of the tubes.CRITICAL STEP Do not disturb bottom phases of the solution when you pipet the upper phase." assertion.
• step description "Centrifuge tubes at 13700 g at 4℃ for 10 min. The white pellet will be visible on the bottom of the tubes.CRITICAL STEP Do not disturb bottom phases of the solution when you pipet the upper phase." assertion.
• step description "Add 700 µl of cold isopropanol to precipitate RNA or DNA and then invert tubes 3-4 times to mix the solution." assertion.
• step description "Add 700 µl of cold isopropanol to precipitate RNA or DNA and then invert tubes 3-4 times to mix the solution." assertion.
• step description "Transfer the upper aqueous phase to a new tube (size= 1.5 ml)." assertion.
• step description "Transfer the upper aqueous phase to a new tube (size= 1.5 ml)." assertion.
• step description "Centrifuge at 13700 g at 4℃ for 10 min.CRITICAL STEP The PH of the extraction buffer is about 8-9, resulting in DNA and RNA precipitation, however, it could result in lower DNA and higher RNA concentrations in case of reducing the PH to around 6-7." assertion.
• step description "Centrifuge at 13700 g at 4℃ for 10 min.CRITICAL STEP The PH of the extraction buffer is about 8-9, resulting in DNA and RNA precipitation, however, it could result in lower DNA and higher RNA concentrations in case of reducing the PH to around 6-7." assertion.
• step description "Add 600 µl of chloroform: isoamyl alcohols (24:1) to each tube, homogenize them by vortexing." assertion.
• step description "Add 600 µl of chloroform: isoamyl alcohols (24:1) to each tube, homogenize them by vortexing." assertion.
• step description "Dissolve the dry pellet in nuclease free water or TE buffer (Ph 8.0). Store DNA at 4 º celcius or -20 º celcius ." assertion.
• step description "Dissolve the dry pellet in nuclease free water or TE buffer (Ph 8.0). Store DNA at 4 º celcius or -20 º celcius ." assertion.
• step description "Dissolve the dry pellet in nuclease free water or TE buffer (Ph 8.0). Store DNA at 4 º celcius or -20 º celcius ." assertion.
• step description "Grind mosquitoes in 1.5 ml eppendorf tube with micropistle in 50-100 µl 1X STE buffer ( 50mM Nacl,50mM Tris- HCL,100mM EDTA, Ph 8.0) along with 100mM sucrose. Add 1X STE buffer to a total volume of 300-500 µl for a single mosquito and 1 ml for mosquito pool like 4,6,8,10 numbers. Then add 1% SDS ,1% Triton -X , 10 µl/ ml Rnase A (20mg/ml), 20 µl/ ml Proteinase K (20mg/ml) and mix it." assertion.
• step description "Grind mosquitoes in 1.5 ml eppendorf tube with micropistle in 50-100 µl 1X STE buffer ( 50mM Nacl,50mM Tris- HCL,100mM EDTA, Ph 8.0) along with 100mM sucrose. Add 1X STE buffer to a total volume of 300-500 µl for a single mosquito and 1 ml for mosquito pool like 4,6,8,10 numbers. Then add 1% SDS ,1% Triton -X , 10 µl/ ml Rnase A (20mg/ml), 20 µl/ ml Proteinase K (20mg/ml) and mix it." assertion.
• step description "Grind mosquitoes in 1.5 ml eppendorf tube with micropistle in 50-100 µl 1X STE buffer ( 50mM Nacl,50mM Tris- HCL,100mM EDTA, Ph 8.0) along with 100mM sucrose. Add 1X STE buffer to a total volume of 300-500 µl for a single mosquito and 1 ml for mosquito pool like 4,6,8,10 numbers. Then add 1% SDS ,1% Triton -X , 10 µl/ ml Rnase A (20mg/ml), 20 µl/ ml Proteinase K (20mg/ml) and mix it." assertion.
• step description "Grind mosquitoes in 1.5 ml eppendorf tube with micropistle in 50-100 µl 1X STE buffer ( 50mM Nacl,50mM Tris- HCL,100mM EDTA, Ph 8.0) along with 100mM sucrose. Add 1X STE buffer to a total volume of 300-500 µl for a single mosquito and 1 ml for mosquito pool like 4,6,8,10 numbers. Then add 1% SDS ,1% Triton -X , 10 µl/ ml Rnase A (20mg/ml), 20 µl/ ml Proteinase K (20mg/ml) and mix it." assertion.
• step description "Wash preserved mosquitoes in sterile distilled water or phosphate buffer saline(PBS) to remove excess alcohol. Fresh mosquitoes can be ground directly." assertion.
• step description "Wash preserved mosquitoes in sterile distilled water or phosphate buffer saline(PBS) to remove excess alcohol. Fresh mosquitoes can be ground directly." assertion.
• step description "Wash preserved mosquitoes in sterile distilled water or phosphate buffer saline(PBS) to remove excess alcohol. Fresh mosquitoes can be ground directly." assertion.
• step description "Wash preserved mosquitoes in sterile distilled water or phosphate buffer saline(PBS) to remove excess alcohol. Fresh mosquitoes can be ground directly." assertion.
• step description "Wash preserved mosquitoes in sterile distilled water or phosphate buffer saline(PBS) to remove excess alcohol. Fresh mosquitoes can be ground directly." assertion.
• step description "Keep the pellet at 37 º celcius for 10 minutes." assertion.
• step description "Keep the pellet at 37 º celcius for 10 minutes." assertion.
• step description "Keep the pellet at 37 º celcius for 10 minutes." assertion.
• step description "Wash the pellet with 70% ethanol ." assertion.
• step description "Wash the pellet with 70% ethanol ." assertion.
• step description "Wash the pellet with 70% ethanol ." assertion.
• step description "Centrifuge at 12,000g for 30 minutes at 4º celcius and then remove the supernatant ." assertion.
• step description "Centrifuge at 12,000g for 30 minutes at 4º celcius and then remove the supernatant ." assertion.
• step description "Transfer the very clear supernatant to a fresh tube , add two fold volume cold isopropanol and keep it for 1 hour at -20º celcius ." assertion.
• step description "Transfer the very clear supernatant to a fresh tube , add two fold volume cold isopropanol and keep it for 1 hour at -20º celcius ." assertion.
• step description "Repeat the above step(5) , then add chloroform:isoamyl alcohol(24:1) and centrifuge at 12,000g for 10 minutes at 4º celcius ." assertion.
• step description "Repeat the above step(5) , then add chloroform:isoamyl alcohol(24:1) and centrifuge at 12,000g for 10 minutes at 4º celcius ." assertion.
• step description "Add equal volume of phenol:chloroform(1:1) ,shake the tube well for 5 minutes and centrifuge at 12,000g for 10 minutes at 4º celcius ." assertion.
• step description "Add equal volume of phenol:chloroform(1:1) ,shake the tube well for 5 minutes and centrifuge at 12,000g for 10 minutes at 4º celcius ." assertion.
• step description "Centrifuge at 12,000g for 10 minutes at 4º celcius .Transfer the supernatant to a fresh tube." assertion.
• step description "Centrifuge at 12,000g for 10 minutes at 4º celcius .Transfer the supernatant to a fresh tube." assertion.
• step description "Lyse for 1 hour 30 minutes at 37º celcius. Gently mix the tube by inverting every 15 minutes." assertion.
• step description "Lyse for 1 hour 30 minutes at 37º celcius. Gently mix the tube by inverting every 15 minutes." assertion.
• step description "After incubation, the tubes were centrifuged at 5000 × g for 10 min at 4°C and the supernatant was gently removed. The pellet is washed two times with 1ml of 70% ethanol and the DNA is pellet by 5000 × g at 4°C for only 5 min. The supernatant is discarded and the pellet is air-dried (10 min). The pellet are allowed to re-suspend in 50 μL of TE (10 mM Tris. HCl pH 8.0; 1 mM EDTA pH 8.0)." assertion.
• step description "After incubation, the tubes were centrifuged at 5000 × g for 10 min at 4°C and the supernatant was gently removed. The pellet is washed two times with 1ml of 70% ethanol and the DNA is pellet by 5000 × g at 4°C for only 5 min. The supernatant is discarded and the pellet is air-dried (10 min). The pellet are allowed to re-suspend in 50 μL of TE (10 mM Tris. HCl pH 8.0; 1 mM EDTA pH 8.0)." assertion.
• step description "After incubation, the tubes were centrifuged at 5000 × g for 10 min at 4°C and the supernatant was gently removed. The pellet is washed two times with 1ml of 70% ethanol and the DNA is pellet by 5000 × g at 4°C for only 5 min. The supernatant is discarded and the pellet is air-dried (10 min). The pellet are allowed to re-suspend in 50 μL of TE (10 mM Tris. HCl pH 8.0; 1 mM EDTA pH 8.0)." assertion.
• step description "After incubation, the tubes were centrifuged at 5000 × g for 10 min at 4°C and the supernatant was gently removed. The pellet is washed two times with 1ml of 70% ethanol and the DNA is pellet by 5000 × g at 4°C for only 5 min. The supernatant is discarded and the pellet is air-dried (10 min). The pellet are allowed to re-suspend in 50 μL of TE (10 mM Tris. HCl pH 8.0; 1 mM EDTA pH 8.0)." assertion.
• step description "Add 2 volumes of cold 95% ethanol and mix gently by inversion. The tubes are incubated at for 45 min in -20°C freezer. It should not be for more time as some remaining phenolics and resin may also precipitate with DNA." assertion.
• step description "Add 2 volumes of cold 95% ethanol and mix gently by inversion. The tubes are incubated at for 45 min in -20°C freezer. It should not be for more time as some remaining phenolics and resin may also precipitate with DNA." assertion.
• step description "Add 2 volumes of cold 95% ethanol and mix gently by inversion. The tubes are incubated at for 45 min in -20°C freezer. It should not be for more time as some remaining phenolics and resin may also precipitate with DNA." assertion.
• step description "After RNAse A treatment, add one volume of chloroform: isoamyl alcohol again and mix by inversions for 5 min. The tube is centrifuged for 10 min at 5000 × g at 4°C. Transfer the clear supernatant into new 2ml tube and add half volume of 5 M NaCl to the sample and mix gently by inversions." assertion.
• step description "After RNAse A treatment, add one volume of chloroform: isoamyl alcohol again and mix by inversions for 5 min. The tube is centrifuged for 10 min at 5000 × g at 4°C. Transfer the clear supernatant into new 2ml tube and add half volume of 5 M NaCl to the sample and mix gently by inversions." assertion.
• step description "After RNAse A treatment, add one volume of chloroform: isoamyl alcohol again and mix by inversions for 5 min. The tube is centrifuged for 10 min at 5000 × g at 4°C. Transfer the clear supernatant into new 2ml tube and add half volume of 5 M NaCl to the sample and mix gently by inversions." assertion.
• step description "Centrifuge the tube for 10 min at 5000 × g at 4°C. Transfer the upper aqueous phase into new 2ml tubes and add 5μl RNAse A (10 mg/mL). Place the tube for 30 min at 37°C." assertion.
• step description "Centrifuge the tube for 10 min at 5000 × g at 4°C. Transfer the upper aqueous phase into new 2ml tubes and add 5μl RNAse A (10 mg/mL). Place the tube for 30 min at 37°C." assertion.
• step description "Centrifuge the tube for 10 min at 5000 × g at 4°C. Transfer the upper aqueous phase into new 2ml tubes and add 5μl RNAse A (10 mg/mL). Place the tube for 30 min at 37°C." assertion.
• step description "Centrifuge the tube for 10 min at 5000 × g at 4°C. Transfer the upper aqueous phase into new 2ml tubes and add 5μl RNAse A (10 mg/mL). Place the tube for 30 min at 37°C." assertion.
• step description "Transfer 1ml of supernatant to each 2ml Eppendorf tubes already containing 1 ml of chloroform: isoamyl alcohol (24:1). Mix supernatant and chloroform: isoamyl alcohol by gentle inversions for 10 min and subsequently place the tube on ice for 10 min." assertion.

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