Isolate 2 rna mini kit

After infection with DENV1–4, the HepG2 cells were collected and the genetic material was extracted through the Isolate 2 RNA mini kit by BioLine, following the protocol according to its manufacturer. The primers used were: DENV 7764 Fwd 5′-CGTCGAGAGAAATATGGTCACACC-3′, DENV 7844 Rev 5′-CCACAATAGTATGACCAGCCT-3′, (no siRNA in this study overlaps with the RT-qPCR primers used), hGAPDH Fwd 5′-TGTTGCCATCAATGACCCCTT-3′, hGAPDH Rev 5′-CTCCACGACGTACTCAGCG-3′, at 0.2 µM, which amplify a region of the NS5 protein [38 (link)]. The total RNA was transcribed to a complementary DNA (cDNA) through SuperScript™ III Reverse Transcriptase by ThermoFisher at 45 °C for 20 min and later amplified through the enzyme from New England Biolabs Luna Universal qPCR Master Mix according to manufacturer’s recommendation with 20 µL. The mix was standardized to a single step. A negative control without RNA template was included for all the RT-qPCR reactions, as well as a control with sample, but without RT and a control without infection.

RPE1 cells were seeded in DMEM (Gibco) supplemented with 10% fetal bovine serum, and one day later the medium was replaced for DMEM without serum. After 2 days cells were infected with the indicated constructs, and 3 days afterwards treated with conditioned media for 15 hours. Neurons were infected at DIV1 with the indicated constructs and treated with 300–400 nM SAG (Enzo Lifesciences) for 24 h, as done before^{61 (link)}. Total RNA was isolated using TRIzol reagent (Life Technologies), phase lock gel tubes and ISOLATE II RNA Mini Kit (Bioline) according to manufacturer’s instructions. The purity and quantity of RNA was assessed on a NanoDrop spectrophotometer, and RNA was reversed transcribed into cDNA with SensiFAST^TM cDNA Synthesis Kit (Bioline). The resulting cDNA was quantified using the SensiFAST™ SYBR No-ROX Kit (Bioline) in the LightCycler 480 (Roche Life Sciences). Analysis was performed using the Advanced Relative Quantification option in the LightCycler 480 software (the Cp value was determined using the second derivative maximum method). The cDNA was quantified in triplicates, and quantification was only considered valid when the Cp values determined did not differ for more than 1 unit among the triplicates. The relative mRNA quantity was normalized to 18 S in RPE1 cells and EEF in neurons. The primers used are provided in Supplementary Table S1.

Akata (EBV+) cells expressing YTHDF2-Myc or YTHDF2 (K281R/K571R/K572R and RRR)-Myc in six-well plates were treated with IgG cross-linking for 12 or 24 h. The cells where then treated with actinomycin D (5 µg/mL) (Cat #A1410, Sigma-Aldrich) to inhibit transcription. The cells were collected at 0, 2, 4, and 6 h after treatment. The total RNA was extracted with an Isolate II RNA minikit (Bioline) and analyzed by quantitative reverse transcription-PCR with specific primers for ZTA, RTA, BALF5, BGLF4, and BLLF1. 18s rRNA was used as control (45 (link)). All primer sequences are listed in Table S1.

RNA was extracted using either the RNA extraction buffers TRIzol (Invitrogen), RNA Bee (Amsbio), and RNAzol RT (Sigma) or an Isolate II RNA minikit (Bioline) according to the manufacturers’ instructions. RT-PCR was performed as described previously (68 (link)). Each primer pair was designed with at least one of them spanning the exon-intron boundary so that mRNA but not genomic DNA was amplified (Table 1) (66 (link), 69 (link)). The cycling parameters were initial denaturation at 94°C for 2 min followed by 25 to 40 cycles of denaturation at 94°C for 10s, annealing at 50 to 55°C for 30 s, and elongation at 68°C for 45 s with a final extension at 68°C for 7 min. PCR band intensity was measured using ImageJ (NIH) or ImageLab 6.0.1 (Bio-Rad) and normalized against an internal control, glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Low-exposure gels were used in image quantification.
Real-time RT-qPCR was performed using 1/50 of the RT reaction mixture in Power SYBR green PCR master mix (Applied Biosystems) and 300 nM sense and anti-sense primers (Table 1). RT-qPCR was run on a StepOnePlus system (Applied Biosystems) using 10 s denaturation at 95°C followed by 40 cycles of 95°C for 15 s and 60°C for 60 s; melting curves were generated using 95°C for 15 s, 60°C for 60 s, and 95°C for 15 s. Data were analyzed by the 2^−ΔΔCT method using GAPDH as an endogenous control.

Mixed stage embryos, 0–12 h after deposition at 18 °C, were dechorionated in bleach, washed in water and 100% ethanol prior to RNA extraction using the ISOLATE II RNA Mini Kit’s protocol (Bioline, UK). The extracted RNA was quantified using Nanodrop (Thermo Fisher Scientific). Library preparation was performed using 1 µg of total RNA and sequencing was performed using Illumina HiSeq 4000 System (2 × 151 bp read length, 40 million reads per sample) by NovogeneAIT Genomics (Singapore). Three biological replicates were sequenced for each condition.

Total RNA was extracted from PCa cells using the Isolate II RNA Mini Kit (Bioline, London, UK) according to standard protocol. RNA concentration and purity were measured using NanoDropTM1000 (Thermo Scientific, BiolaB, Scoresby, VIC, Australia). A total of 1 μg of RNA was reverse transcribed to cDNA using SensiFast^TM cDNA synthesis kit (Bioline, GmbH, Luckenwalde, Germany). The cDNA was diluted to 100 μL before using it as a template for PCR reaction.

RNA was extracted using Isolate II RNA Mini Kit (Bioline) and 1 µg of total RNA was reverse transcribed into cDNA using Maxima First Strand cDNA kit (Thermo Scientific). Primers are listed in Supplementary Table 1. Real-time quantitative PCR amplification was performed using a 96-well plate system (Licor). Gene expression was normalized using the ΔΔ Ct method, using RPL13 as a housekeeping gene.