Random primer hexamers

For the analysis of the presence of ZIKV in placental samples, approximately 25 mgs of tissue were homogenized using the Molecular Grinding Resin (G-Biosciences, St. Louis, MO, USA) and subsequently processed with NuceloSpin RNA II extraction kit for total RNA extraction according to the manufacturer’s protocol. Complementary DNA (cDNA) was obtained with the SuperScript II Reverse Transcriptase kit (Invitrogen Life Technologies, Carlsbad, CA, USA) by using Random Primer Hexamers (Invitrogen Life Technologies) and following the manufacturer’s instructions. ZIKV RNA levels were assessed by quantitative RT-PCR using the iTaq Universal Probes Supermix (Bio-Rad, Reinach, Switzerland) and the Rotor Gene 6000 thermocycler (Corbett Research, Sydney, Australia) as previously described [25 (link)]. As positive controls, placental homogenates were spiked with different inocula of ZIKV strain PRVABC59.

Total RNA was extracted from cell-culture supernatants using the QIAamp^® Viral RNA mini kit (Qiagen, Hilden, Germany) and used for the synthesis of the first cDNA strand using SuperScript III reverse transcriptase and random primer hexamers (Invitrogen, Life Technologies, Carlsbad, CA, USA). The second cDNA strand was obtained using the NEBNext^® Ultra™ II Non-Directional RNA Second Strand Synthesis Module kit (New England BioLabs, Hitchin, UK). Library preparation was then completed with the NEBNext^® Ultra™ II RNA Library Prep Kit for Illumina (New England BioLabs). After quality validation on the 2100 BioAnalyzer (Agilent Technologies, Santa Clara, CA, USA), the library was sequenced to obtain 2 × 150 bp paired-end reads on an Illumina NovaSeq 6000 sequencer (Integragen, Ivry, France).

One microgram of total RNA or 100 ng of size-extracted RNA (20-150 nt) from all experimental conditions were treated with DNase I (Thermo Scientific, Waltham, MA, USA) to digest any leftover DNA, according to the manufacturer’s protocol. Samples were then reverse transcribed into cDNA with SuperScript™ IV One-Step RT-PCR System (Invitrogen, Carlsbad, CA, USA) and random primer hexamers (Thermo Scientific, Waltham, MA, USA). cDNA samples were treated with RNase H (NEB, Ipswich, MA, USA) to hydrolyze leftover RNA. qPCR was done using GoTaq^® qPCR Master Mix (Promega, Madison, WI, USA), 50-fold diluted cDNA, and 500 nM to 1 uM of each primer. qPCR reactions were prepared by the CAS-1200 Corbett robot (Corbett Robotics, San Francisco, CA, USA) and were carried out using the Rotor Gene 6000, with suggested standard cycling conditions for gyrA and rpoS, and FAST cycling conditions for sRNA validation (Promega, Madison, WI, USA). 5S and recA were used as an internal control for the normalization of gene expression. The samples were run in duplicates. The 2^−ΔΔCT method was used to calculate the fold-change relative to the control [65 (link)]. The mean log₂ fold-change and standard error of the mean were computed. Oligonucleotides used for RT-qPCR are provided in Supplementary Table S5.