Iscript advanced kit

mRNA was extracted from snap‐frozen 16h‐stimulated moDCs using a NucleoSpin® RNA Plus kit (Macherey‐Nagel, Germany) in combination with the rDNAse set (Macherey‐Nagel, Germany) to remove contaminating DNA. cDNA was synthesized using an iScript™ advanced kit (Bio‐Rad, USA) according to the manufacturer's protocol in the PTC‐100 Programmable Thermal Controller (MJ research, PTC‐100). cDNA and mRNA samples were stored at ‐80°C. Quantitative analysis was conducted on a CFX96 Real‐Time C1000 Thermal Cycler detection system with the use of an IQ™ SYBR® Green Supermix, according to manufacturer's protocol (both from Bio‐Rad). Primers for the genes of interest are listed in Table 1. Primers (produced by Biolegend, USA) were designed with Primerblast (NCBI, USA) and Beacon Designer (Premier Biosoft International, USA). Primers were validated based on the efficiency and R². Primer specificity was tested by running the products on a 2% agarose gel (70 minutes; 110V). The expression of the genes of interest was determined using 12.5ng cDNA and SYBR green (Bio‐rad, USA). The RT‐PCR protocol consists of 3 min at 95°C followed by 10 s at 95°C and 30 s at the optimal primer annealing temperature as determined beforehand. The samples were measured in duplo and mean Ct values were used to determine the relative expression using the Pflaff method and β‐actin as a reference gene.

Whole brain, kidney, spleen, thymus, BM, heart, and small intestines from three independent biological replicates from Adar1^+/+Ifih1^-/- and Adar1^E861A/E861AIfih1^-/- were used for mmPCR-seq. Following tissue homogenization, RNA was isolated using TRIsure reagent (Bioline) and Direct-Zol columns (Zymo Research). cDNA was synthesized using iScript Advanced Kit (BioRad) using 1.2–7.5 μg RNA per tissue. A total of 200 ng of cDNA was used for mmPCR-seq targeting 557 loci containing 11,103 known A-to-I editing sites [48 (link)]. A-to-I editing frequency was assessed using the criteria: minimum coverage > 100 (in 2/3) of WT (Adar1^+/+Ifih1^-/-) and deficient (Adar1^E861A/E861AIfih1^-/-). For a given site, where a replicate had ≤ 100 reads that measurement is regarded as a missing value. All editing sites with editing ≤ 0.01 were removed, leaving 413–808 sites per tissue. Sites were ADAR1-specific targets if the editing level measurements between the WT (Adar1^+/+Ifih1^-/-) replicates and the editing-deficient (Adar1^E861A/E861AIfih1^-/-) replicates were significantly different (P < 0.1, ANOVA) and the average editing levels between WT and editing-deficient samples differed by at least 5% [48 (link)].