Adar-mediated A-to-I editing is required for establishment of embryonic body axes in zebrafish

Adar-mediated A-to-I editing is required for establishment of embryonic body axes in zebrafish. To assess the effect of Adar KD and OE, uninjected wild-type and embryos injected with 1 ng of adar MO or 50 pg of adar mRNA were kept until desired developmental stage: 128-cell or 50% epiboly. Two replicates of 20 pooled embryos from each time point were isolated for RNA extraction. For the analysis at 12 hpf, a separate batch of uninjected control, as well as Adar KD and OE samples were generated in three replicates. Parental DNA was extracted from one individual male and female fishes from tail-fin clipping, together with RNA from their offspring at 16-cell, 3.5 hpf, and 5.3 hpf stages. For each time-point, at least 20 embryos were pooled for RNA extraction. To assess the effect of Adar KD and OE, uninjected wild-type and embryos injected with 1 ng of adar MO or 50 pg of adar mRNA were kept until desired developmental stage: 128-cell or 50% epiboly. Two replicates of 20 pooled embryos from each time point were isolated for RNA extraction. For the analysis at 12 hpf, a separate batch of uninjected control, as well as Adar KD and OE samples were generated in three replicates. Total RNA was extracted using TRIzol LS (Thermo Fisher Scientific, USA) and cleaned up on the Qiagen Rneasy Mini column (Qiagen, USA). Quality control of extracted RNA was performed using the 2200 TapeStation system from Agilent Technologies (USA). To avoid the bias caused by cytoplasmic polyadenylation during early embryonic stages, total RNA was rRNA-depleted using Ribo-Zero Magnetic Gold Kit (Human, Mouse, Rat; Epicenter). cDNA synthesis for Next-Generation Sequencing (NGS) was performed by SMARTer Stranded RNA-seq kit  (Clontech Laboratories, USA) as recommended by the manufacturer. Paired-end sequencing (2 × 75 bp reads) was performed with NextSeq 500 (Illumina, USA).