Oligo dt 18 primer

RNA was extracted from hippocampus, cortex and brainstem using peqGOLDTriFastTM (PeqLab, 30-2040, Erlangen, Germany) reagent according to the manufacturer’s instructions. RNA was treated with DNaseI (Life Technologies, EN0521) to remove genomic DNA contamination. This procedure was followed by cDNA synthesis using Superscript III-reverse transcriptase (Life Technologies, 18,080,085) and Oligo (dT)₁₈-Primers (Thermo Scientific, SO132) at 50 °C for 1 h. cDNA was used as PCR template in an 1:10 dilution and each sample run in triplicate. Quantitative PCR was performed on StepOnePlus™ Real-Time PCR System (Applied Biosystems) using Express SYBR GreenER qPCR Supermix Universal (Life technologies, 1,178,401 K), 0.2 μM primer each on the DNA (primers as published by us before) with the following cycle conditions: primary denaturation at 95 °C for 3 min at 95 °C, 35 cycles with 30s at 95 °C, 30s at 60 °C and 30s at 72 °C followed by fluorescence measurement. Absolute quantification was performed for every single gene with three technical repeats per sample. Serial dilutions of plasmid controls with known molecule concentrations were used as a positive control and to generate standard curves. Expression of target genes were normalized using 36B4 (large ribosomal protein P0, RPLP0) as reference gene.

RNA was extracted with TRIzol (Invitrogen) and treated with DNaseI-RNase-free (Thermo Scientific). Reverse transcription reactions were performed with oligo(dT)₁₈ primers (Thermo Scientific) and RevertAid reverse transcriptase (Thermo Scientific), according to manufacturer’s instructions. RT material (1/20) was used as template for qPCR with SYBR green (Biotool) on a 7500 Real time PCR System (Applied Biosystems). Primer sequences are described in S2 Table. Quantification was carried out as previously described [56 (link)].

Total RNA was isolated using the miRNeasy kit (Qiagen, Venlo, the Netherlands) following the manufacturer’s instructions and included a DNase treatment on the column for 30 min at room temperature. RNA concentrations were determined using the Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Bleiswijk, the Netherlands). 1–3 μg RNA was reverse transcribed with the RevertAid H Minus First Strand cDNA synthesis kit and Oligo(dT)18 primers (both Thermo Fisher Scientific, Bleiswijk, the Netherlands), following the instructions of the manufacturer. cDNA was treated with 2 units of RNaseH (Thermo Fisher Scientific, Bleiswijk, the Netherlands) for 20 min at 37 °C. RT-qPCR analysis was performed with the CFX96 system (Bio-Rad, Veenendaal, the Netherlands) using iQ SYBR Green Supermix (Bio-Rad, Veenendaal, the Netherlands), 0.5 pM of each primer (sequences are listed in Supplementary Table 3), and 1:5 or 1:50 diluted cDNA. The following cycling conditions were used: an initial denaturation step at 95 °C for 3 min followed by 40 cycles of 10 seconds at 95 °C and 30 seconds at primer Tm. A melting curve analysis from 65 °C to 95 °C (temperature increments of 0.5 °C) was performed to determine the specificity of each reaction. Data were analyzed with Bio-Rad CFX Manager version 3.1 (Bio-Rad, Veenendaal, the Netherlands) and normalized to the housekeeping genes Gapdh and Rpl13a.

LCMV (Armstrong) was obtained from D. Pinschewer (European Virus Archive Global). For batch production, hamster BHK-21 cells were infected at a multiplicity of infection of 0.01, and the virus-containing supernatant was collected 48 h after infection. Mice were infected by intraperitoneal injection of 2 × 10⁵ plaque-forming units. Detection of LCMV in the spleen was performed by quantitative PCR with reverse transcription (RT–qPCR). Total RNA was isolated by TRIzol LS (Invitrogen, 10296010), and in-column DNase digestion was performed using an RNA Clean & Concentrator kit (Zymo Research), according to manufacturers’ instructions. RNA was stored at −80 °C or transcribed immediately using RevertAid reverse transcriptase (Thermo Fisher Scientific, EP0442) with oligo(dT)18 primers (Thermo Fisher Scientific, SO131) according to the manufacturer’s instructions. RT–qPCR was performed using LightCycler 480 SYBR green I master mix (Roche, 04887352001) and a LightCycler 480 II machine (Roche). All samples were measured in triplicates. The LCMV titers were quantified against a standard curve from cloned S-segment of LCMV in pBlueScript vector (Supplemetary Table 6). The quality of isolated RNA was tested by RT–qPCR analysis of an endogenous reference gene Eef1a1 (Supplemetary Table 6).

For analyzing gene expression, cells were isolated with TRIzol reagent (Thermo Fisher Scientific, Carlsbad, CA, USA). RNA isolation, cDNA synthesis, and quantitative PCR were performed as previously described [33 (link),34 (link),80 (link)]. Briefly, 1-bromo-3-chloro-propane (Sigma-Aldrich, St. Louis, MO, USA) and centrifugation were used for the separation of RNA. Cleaning of RNA was performed with an RNA Clean & Concentrator-5 kit (Zymo research, Freiburg, Germany). The quality and quantity of the RNA were measured with Nanodrop OneC (Thermo Fisher Scientific, Carlsbad, CA, USA). SuperScript IV Reverse Transcriptase (Thermo Fisher Scientific, Carlsbad, CA, USA) and Oligo(dt)18 primers (Thermo Fisher Scientific, Carlsbad, CA, USA) were used for cDNA synthesis. Quantitative PCR was performed with Luminaris Color HiGreen qPCR Master Mix (Thermo Fisher Scientific, Carlsbad, CA, USA) according to the manufacturer’s protocol and analyzed with qTOWER3 (Analytik Jena, Jena, Germany). Primer design was performed as previously described [33 (link),34 (link),80 (link)]. The quality and specificity of the primers were analyzed using melting curves and agarose gel electrophoresis. Dilution series of cDNA were used to calculate primer efficiency. Table 1 contains all information on the used primers. RPL22 and TBP were used as reference genes for data analysis according to the ∆∆CT method.

Total RNA was isolated from cell lysates using RNeasy mini kit (Qiagen) and reverse transcribed with SuperScriptIII reverse transcriptase (18080-044, Invitrogen) using oligo(dT)₁₈ primers (Thermo Scientific) following the manufacturers' instructions. Quantitative PCR was performed using SYBR Green I Master Mix (Roche) on a LightCycler 480 II instument (Roche). Primers are listed in Supplementary Table 1. All quantitations were normalized to endogenous GAPDH. Relative changes in gene expression were quantified using the 2^−ΔΔC_T method37 (link).