E pap

5 μl of TURBO DNase treated RNA was polyadenylated with E. coli PAP (E-PAP, ThermoFisher Scientific) in 20 μl reactions, containing 1x reaction buffer, 2.5 mM MnCl₂; 0.4 U E-PAP and 0.8 U RiboLock RNase inhibitor (ThermoFisher Scientific) for 30 min at 30°C. Reactions were then purified using a PureLink micro RNA purification kit (Ambion) and eluted using 22 μl RNase-free water. PureLink eluates were rRNA depleted using a down-scaled reaction of the Ribo-Zero Gold rRNA Removal Kit for Yeast (Illumina). That is, 18 μl of RNA was incubated with 2 μl of reaction buffer, 2 μl of removal solution and 8 μl of water for 10 min at 65°C, and put to RT for 5 min. This reaction was cleared using 65 μl of rRNA magnetic beads for 5 min at RT and 5 min at 50°C. Final samples were precipitated using NaOAc and ethanol with 20 μg of glycogen as carrier and resuspended in 10 μl of RNA storage solution.

3′ Rapid Amplification of cDNA Ends (RACE) was performed by poly-adenylating the ZIKV RNA using E-PAP (Thermo Fisher Scientific) followed by cDNA synthesis and PCR using FirstChoice RLM-RACE Kit (Thermo Fisher Scientific). The sequence of the ZIKV-specific primer used at the PCR step was 5′-AGAGTGTGGATTGAGGAGAACGAC-3′. PCR products were then cloned and sequenced with Sanger technology prior to incorporation into the respective consensus genome sequence. Sequences obtained by 3′ RACE extended those obtained by NGS by approximately 70 nucleotides.

Total RNA from HOG and M17 cells were treated for poly (A) tailing and with RNase R as described in the published method with modifications [32 (link)]. In brief, 3 μg of total RNA was subjected to poly (A) tailing in a 50 μL reaction using the poly (A) tailing kit (Thermo Fisher AM1350) following manufacturer’s instructions; 2 μL E-PAP and 40 U RNase inhibitor (Thermo Fisher Scientific N8080119) were also added to the reaction and incubated at 37 °C for 1 h. First, the RNAs were purified by the RNA Clean & Concentrator-25 (Zymo R1018) kit and eluted in 25 μL nuclease-free water. The RNAs were then treated with 5 U RNase R in 30 μL reactions which contained 25 μL of all RNA samples from A-tailing reaction, 3 μL 10× RNase R Buffer (0.2 M Tris–HCl (pH 8.0), 1 mM MgCl_2, and 1 M LiCl) and 1 μL RiboLock RNase inhibitor (40 U/ μL) (Thermo Fisher Scientific EO0381). According to the manufacturer’s instructions, reactions were purified with RNA Clean & Concentrator-25 (Zymo Research R1018), and the RNA was eluted in 30 μL nuclease-free water. Then, the amount of RNAs (in 30 μL nuclease-free water) was used to prepare rRNA depleted RNA-seq library following the KAPA RNA HyperPrep Kit with RiboErase (HMR).