Direct zol columns

Larval zebrafish islets were isolated as previously reported^{77 (link)}. Briefly, Tg(gcga:EGFP) larvae were euthanized in ice-cold water and suspended in a solution of HBSS with 50 μg/ml Liberase DH (5401119001, Sigma), lightly crushed with a pestle and incubated at 37 °C for 2 min. The entire content was quickly placed in a 10 cm petri dish containing RPMI (GIBCO) with 10% Fetal Bovine Serum (Atlanta Biologicals). Islets were picked manually under a fluorescence stereomicroscope and placed into a 6 cm dish with RPMI. This process was repeated to limit extraneous tissue. At least 50 islets were pooled for RNA extraction.
RNA of larvae and mouse islets was isolated using TRIZOL reagent (Thermo Fisher Scientific) with Direct-Zol columns (Zymo Research) and concentrated using RNA Clean and Concentrator-25 columns (Zymo Research). RNA was reverse transcribed using Superscript III (Thermo Fisher Scientific) with an oligo-dT primers. The cDNAs were subjected to PCR on a BioRad CFX96 machine (Bio-Rad Laboratories, CA, USA) and amplicons were detected using SYBR green (Bio-Rad). The levels of expression were determined by the Pfaffl method to compare Ct values of ef1a (zebrafish) or Actb (mouse). All the primers used in this study are listed in Supplementary Table 3.

Worm aliquots were thawed and 500 μL of Trizol LS (Life Technologies, 10296–028) was added and mixed vigorously. Next, we employed six freeze-thaw cycles to dissolve the worms: tubes were frozen in liquid nitrogen for 30 seconds, thawed in a 37°C water bath for 2 minutes, and mixed vigorously. Following the sixth freeze-thaw cycle, 1 volume of 100% ethanol was added to the samples and mixed vigorously. Then, we added these mixtures onto Direct-zol columns (Zymo Research, R2070) and manufacturer’s instructions were followed (in-column DNase I treatment was included).

Mouse tissues from three independent biological replicates from age-matched and sex-matched C57BL/6, Adar1^+/+Ifih1^-/-, and Adar1^E861A/E861AIfih1^-/- mice were homogenized in Trisure reagent using IKA T10 basic S5 Ultra-turrax Disperser. Brain tissue was isolated from the pups collected at the day of birth and snap-frozen, then homogenized in Trisure reagent using IKA T10 basic S5 Ultra-turrax Disperser. RNA was extracted using Direct-Zol columns (Zymo Research) as per manufacturer’s instructions. Complementary DNA (cDNA) was synthesized using Tetro cDNA synthesis kit (Bioline). Real-time PCR was done in duplicate with Brilliant II SYBR Green QPCR Master Mix (Agilent Technologies) and primers from IDT (Additional file 3: Table S2). All primers were optimized to have equal efficiency (100+/-10%) before use. Ppia was used as a reference gene for relative quantification using the ∆Ct method. RNA was obtained from fetal brain from E12.5 Adar1^E861A/E861A embryos and processed as described above [19 (link)].