GSM9632985: Day 1 Controls_R4; Danio rerio; RNA-Seq
Pooled embryos were transferred to 1.5 mL twist-cap microtubes (NextAdvance, TUBE1R5-S) and euthanized on ice. After removing excess water, 200 μL of 1× RNAshield (Zymo Research) was added following 15 min on ice. Samples remained at room temperature for up to 15 min before storage at −20 °C. Four pools of eight embryos were collected per treatment group and corresponding controls. For RNA isolation, tubes were thawed at 32 °C for 30 min, followed by addition of 100 μL of 0.5-mm zirconium silicate beads (BioSpec). Homogenization was performed using a Bullet Blender Storm Pro (NextAdvance) at speed 10 for 5 min. Samples were centrifuged at 12,000 × g for 5 min, and 250 μL of supernatant was aliquoted into two new plates (totaling 500 μL). RNA was extracted using the KingFisher Apex automated system with the Zymo Quick RNA MagBead Kit and eluted in 50 μL RNase/DNase-free water. RNA integrity was assessed using an Agilent Tapestation 4200, while RNA quantity was measured using ThermoFisher QuantIT; only samples with RIN > 8 were used for sequencing. In total, 48 samples, including 8 control replicates, were prepared for RNA-seq. mRNA sequencing was performed on four biological replicates per treatment. Library preparation used PrepX Robotic RNA PolyA enrichment combined with NEBNext UltraII Directional RNA Library Prep / Illumina Stranded mRNA workflow. Samples were distributed across three P3 flow cells (1.2 billion reads/run) and one P2 flow cell (400 million reads/run) and sequenced on an Illumina NextSeq 2000 platform (100 bp single-end), yielding approximately 20 million reads per sample.