GSM9127836: TPP-2SK_S36_2; Oryzias latipes; RNA-Seq
At 14 dpf, larval medaka were euthanized in ice-cold embryo rearing medium (ERM), and pooled in microcentrifuge tubes to obtain 15–20 individuals per RNA sample. Re-sidual water was removed, and Leibovitz's L-15 medium (Corning, Mediatech, Inc., Manassas, VA, USA) supplemented with 10% fetal bovine serum (FBS; Atlanta Biolog-icals) was added. To isolate axial skeletal tissue, hatched larvae were passed multiple times through a 20–22-gauge hypodermic needle attached to a 3-mL syringe (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). The resulting tissue suspension was filtered through 105-μm nylon mesh (Component Supply Co., Fort Meade, FL, USA) and transferred to fresh L-15/10% FBS medium. Under a dissecting microscope (Nikon SMZ1500, Nikon Instruments, Inc., Melville, NY, USA), axial tissue was carefully ex-amined and trimmed to remove any remaining craniofacial or abdominal tissue. Cleaned samples were immediately flash-frozen in liquid nitrogen. Tissues were homogenized in TRI Reagent® (Ambion®, Life Technologies, Carlsbad, CA, USA) using a handheld BioVortexer (Thomas Scientific, Swedesboro, NJ, USA), and total RNA was extracted following the manufacturer's protocol. RNA concentration was determined using the Qubit® RNA HS Assay Kit (Invitrogen) with the Qubit® 3.0 Fluorometer Illumina RNA library preparation and sequencing were performed at the NC State Genomic Sciences Laboratory (Raleigh, NC). Total RNA was extracted from DMSO- and TPhP-treated embryos/larvae (n= 3-4 technical replicates of 3-4 biological replicates of 15-20 pooled larvae), and assessed for quality, concentration, and integrity using RIN > 9) an Agilent 2100 Bioanalyzer with an RNA 6000 Nano Chip (Agilent Technologies, Cary, NC). From 1 µg of total RNA, messenger RNA (mRNA) was purified using oligo-dT magnetic beads from the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, Ipswich, MA). Complementary DNA (cDNA) libraries were generated using the NEBNext Ultra Directional RNA Library Prep Kit and NEBNext Multiplex Oligos for Illumina, following the manufacturer's instructions. In brief, purified mRNA was chemically fragmented and reverse-transcribed into first-strand cDNA using random primers. Second-strand synthesis incorporated dUTPs to retain strand specificity. The resulting double-stranded cDNA was purified, end-repaired, and adenylated prior to adapter ligation. Size selection was performed using AMPure XP beads (Beckman Coulter, USA) to isolate library fragments between 400–550 bp (including adapters). Libraries were then PCR-amplified to enrich fragments and incorporate unique index barcodes. Final libraries were purified and assessed for size distribution and concentration using an Agilent 2200 TapeStation with a High Sensitivity DNA chip and a Qubit fluorometer (Thermo Fisher, Raleigh, NC). Indexed libraries were pooled in equimolar amounts and sequenced on an Illumina HiSeq 2500 platform using a 125 bp single-end sequencing kit. Base calling was conducted with Real Time Analysis (RTA) software to produce raw BCL files, which were subsequently demultiplexed into FASTQ files for downstream analysis.