Random hexamers

Cells were seeded in 35 mm dishes with a density of 5 × 105 cells/dish and synchronized by fresh medium addition. Cells were collected every three hours, in triplicates and the pellets were stored at −80 °C. Total RNA was isolated using the RNeasy extraction kit (Qiagen), including DNase digestion, according to the manufacturer’s instructions. Cells were lysed with 350 µl RLT buffer and the lysate was homogenized. Total RNA concentration was determined using NanoDrop® ND-1000 UV-Vis Spectrophotometer and stored at −80 °C °C. 1 µg of total RNA was reverse-transcribed to cDNA with M-MLV reverse transcriptase (Invitrogen), random hexamers (Eurofins MWG Operon) and dNTPs Mix (Thermo Fisher).

The zebrafish genome, contains at least 7 vtg genes (42 (link)) and there is evidence that their expression varies over different life stages (42 (link)–44 (link)) which may have a bearing on life stage sensitivities to vtg mRNA induction. We chose vtg1 mRNA as previous work has suggested that it is the most ubiquitously expressed vtg gene transcript (45 (link)). Whole body samples (for 21, 30 and 60 dpf fish) and liver samples (90 dpf) for vtg and gfp mRNA expression analysis were immediately snap-frozen in liquid nitrogen upon collection and stored at −80 °C until use. Total RNA was extracted from all samples using Tri Reagent (Sigma), according to the manufacturer’s instructions. The amount of RNA was quantified using a NanoDrop 1000 spectrophotometer and RNA purity determined through the measurement of the A260/A280 and A260/A230 ratios, respectively. Reverse transcription was subsequently carried out by incubating 1 µg RQ1 DNase treated (Promega, Southampton, UK) total RNA with 5 mM random hexamers (Eurofins MWG Operon. Ebersberg, Germany), 10 mM dNTPs and 1 µl MMLV-Reverse transcriptase (Promega) in the appropriate buffer, following the manufacturer’s instructions.

Cells were seeded in 35 mm dishes with a density of 5 × 10⁵ cells/dish and synchronized by fresh medium addition. Cells were collected every three hours, in triplicates and the pellets were stored at −80 °C. Total RNA was isolated using the RNeasy extraction kit (Qiagen), including DNase digestion, according to the manufacturer’s instructions. Cells were lysed with 350 µl RLT buffer and the lysate was homogenized. Total RNA concentration was determined using NanoDrop® ND-1000 UV-Vis Spectrophotometer and stored at −80 °C. 1 µg of total RNA was reverse-transcribed to cDNA with M-MLV reverse transcriptase (Invitrogen), random hexamers (Eurofins MWG Operon) and dNTPs Mix (Thermo Fisher).

Five μg of total RNA were circularized with 40 U of T4 RNA ligase (New England Biolabs), following the manufacturer's instructions. Circularized RNA was purified using the RNA Clean & Concentrator Kits (Zymo Research®, California, USA). The first strand complementary DNA (cDNA) synthesis was done for 3 h at 40°C using 400 U of M-MLV reverse transcriptase (Fermentas), 8 mM of random hexamers (Eurofins), 1× M-MLV buffer, 0.5 mM dNTPs and 40 U of Riboblock RNase inhibitor (Fermentas). The obtained cDNA was diluted four times, and 5 μl of the obtained cDNA solution was used for PCR amplification with divergent primers. The primers used for mapping precursor transcripts are listed in Supplementary Table S1. Amplified PCR products were gel purified, cloned into pCR2.1^®-TOPO^® TA vector (ThermoFisher Scientific) and inserts of independent recombinant plasmids were sequenced after E. coli transformation.