GSM5268198: DENV_1to500_rep2; Dengue virus 1; OTHER
2 µl of 10× T4 polynucleotide kinase (PNK) buffer and 2 µL of T4 PNK were added to 14 µL of each vRNA sample. Reaction mixtures were incubated for 4 h at 37 °C. Then, additional 0.5 µL of T4 PNK and 2 µL of 10 mM ATP were added, and the mixture reactions were incubated for 1 h at 37 °C. The final volume of the mixtures was adjusted to 50 µL by adding nuclease-free water. Following, high-quality vRNA samples were prepared with an RNA Clean & Concentrator kit (RNA Clean & Concentrator-5, Zymo Research). Briefly, 100 μL of RNA binding buffer were added to each 50 μL of vRNA sample. Then, an equal volume of ethanol 100% was added and mixed. Samples were transferred to a column in a collection tube and centrifuged for 30 s at 12,000 × g. The flow-through was discarded and 400 μL of the second RNA buffer were added. Viral RNA bound to the columns were washed twice. A dry spin was made to completely remove the washing buffer. Columns were transferred into RNA-free tubes and the vRNA was eluted by adding 8 μL of RNase-free water to the column matrix. After 1 min incubation, columns were centrifuged for 30 s at 12,000 × g. The recovered vRNA was double eluted and samples were storage at -80 °C. Library preparation was made with a NEBNext Multiplex Small RNA Library Prep Set for Illumina kit (New England Biolabs), with small modifications to the protocol. 1 μL of 3' SR Adaptor was added to 6 μL of the high-quality vRNA samples prepared, in nuclease-free PCR tubes. Reaction mixtures were incubated in a preheated thermal cycler for 2 min at 70 °C, and then transferred to ice. 10 μL of 3' Ligation Reaction Buffer and 3 μL of 3' Ligation Enzyme Mix were added to each reaction mixture. Samples were incubated in a thermal cycler for 1 h at 25°C. To prevent the formation of dimers of the remaining 3' SR Adaptor, 4.5 μL of nuclease-free water and 1 μL of SR reverse transcription (RT) Primer for Illumina were added, and samples were incubated for 5 min at 75 °C, 15 min at 37 °C and 15 min at 25 °C. For the ligation of the 5' SR Adaptor, an adaptor mix was prepared. For that, 5' SR Adaptor was incubated in a thermal cycler at 70 °C for 2 min, and then immediately transferred to ice. Following, 1 μL of the denatured 5' SR Adaptor was mixed with 1 μL of 5' Ligation Reaction Buffer and 2.5 μL of 5' Ligation Enzyme Mix. The adaptor mix was added to the samples with 3' SR Adaptor and incubated in a thermal cycler for 1 h at 25 °C. To perform the RT of the adaptor ligated vRNA samples, 30 μL of these samples were mixed in nuclease-free tube with 8 μL of First Strand Synthesis Reaction Buffer, 1 μL of Murine RNase Inhibitor and 1 μL of ProtoScript II Reverse Transcriptase. Reaction mixtures were incubated at 50 °C for 60 min. Then, RT reactions were inactivated at 70 °C for 15 min and stored at -20 °C. A large-scale PCR amplification was performed using 12 PCR cycles, the number of cycles identified as enough to construct a library. For that, 10 μL of the cDNA were mixed, on ice, with 2.5 μL of F-primer (SR primer), 2.5 μL of R-primer (with different barcodes for each sample), 5 μL of nuclease-free water and 20 μL of 2X Q5 Master Mix. The barcoded cDNA samples were normalized to 1000 ng/mL and sent to next generation sequencing on the Illumina platform.