Iscript master mix

Following incubation in the noncontact coculture system in mCDM (pH 6.5) containing 1.0 mM l-methionine for 8 or 16 h at 37°C, bacterial cells in 27 mL of each monocultured and cocultured bacterial solution were collected by centrifugation (8,000 × g for 7 min at 4°C). The cells were then resuspended in 3 mL of RNAprotect Bacteria Reagent (Qiagen, Hilden, Germany). After incubation for 5 min at room temperature, 1 mL of the bacterial solution was collected and immediately frozen, then treated with 20 µL of proteinase K (Qiagen) and 200 µL of 15 mg/mL lysozyme at 55°C, and subjected to centrifugation at 1,000 rpm for 15 min. RNA isolation was performed with 1 mL of TRIzol reagent (Life Technologies Corp., CA, USA) and an RNeasy kit (Qiagen). cDNA synthesis along with the removal of genomic DNA was performed using iScript master mix (Bio-Rad, CA, USA) according to the manufacturer’s instructions. Real-time PCR assays were performed with a KAPA SYBR Fast kit (KAPA Biosystems, MA, USA), following the supplied protocol. Designed primers are shown in Table S1. To determine gene expression, a comparative Ct method was used.

For RNA extractions, we used the illustra RNAspin Mini Kit (GE Healthcare Life Sciences, Little Chalfont, UK) or the RNeasy Mini Kit (QIAGEN, Hilden, Germany). For complementary DNA conversion, we used iScript master mix (Bio-Rad Laboratories, Hercules, CA, USA), and for quantitative PCR (qPCR) reactions, we used SsoAdvanced SYBR Green Master Mix (Bio-Rad Laboratories) on a CFX384 thermocycler (Bio-Rad Laboratories), according to the manufacturer’s instructions. Expression data were normalized to RPLP0. Primer sequences are available in Additional file 2.

Prior to treatment, cells were starved for 2 hr in serum free growth media. Cells were then treated with 2, 0.5, 0.1, 0.05, or 0.01 ng/mL TGF-β1 or BSA vehicle control for 2 hr. For inhibitor assays, LY2109761 (Selleck Chemicals, Houston, TX, USA), a pharmacological inhibitor of TGF-β receptor type I/II, was dissolved in DMSO at a concentration of 2 mM. Cells were treated with 2 µM LY2109761 or DMSO vehicle control. Cells were lysed and RNA isolation was performed using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol, homogenized using Qiashredder columns (Qiagen), and on column DNAse digestion. Quantification of total RNA was performed using a NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA). Total RNA (0.5 µg) was reverse transcribed to cDNA using iScript master mix (Bio-Rad, Hercules, CA, USA). Equal amounts of cDNA per condition, SYBR green master mix (Applied Biosystems, Foster City, CA, USA), and primer probe sets (see methods Table) were detected with a StepOnePlus RT- PCR system (Applied Biosystems). The mRNA fold changes were determined using the ΔΔCT method normalized to the 18S endogenous control [36 (link)].

For cellular messenger RNA (mRNA) analysis, RNA was extracted from the cell lines using an RNeasy Mini kit (Qiagen, Hilden, Germany), following the manufacturer's instructions. RNA samples were treated with RNase-free DNase (Qiagen) and complementary DNA (cDNA) was generated from total RNA (500 ng per sample) using iScript Mastermix (Bio-Rad), according to the manufacturer's instructions. Quantitative PCR (qPCR) was performed with SYBR Green PCR MasterMix (Bio-Rad) using a Roche LightCycler 480(Roche Diagnostics, Basel, Switzerland).

Human microglial cell line was infected with ZIKV at MOI of 1 in cell medium containing metabolic inhibitors such as 5-fluorouracil (ab142387; Abcam, Cambridge, UK) and floxuridine (4659; Tocris, Bristol, UK) in cell medium containing 1 and 10 μM of each drug or 1% (vol/vol) dimethylsulfoxide (DMSO) as a control. After 48 h post infection, cellular RNA was extracted using Trizol and cDNA was synthesized by iScript Mastermix (Bio-Rad), as per the manufacturer's instructions. ZIKV RNA was quantified by using specific primers by SYBR Green PCR Master Mix (Bio-Rad), using a Roche LightCycler 480. Further, immunocytochemistry was also performed in microglial cells for the detection of ZIKV infection using flavivirus group antigen-specific antibody (Millipore).