title: RNA-Seq of Danio rerio: Larvae bone
design: Eukaryotic mRNA sequencing by second-generation high-throughput sequencing technology platforms was used to analyze all mRNA transcribed by specific tissues or cells at a specific time point. The RNA samples used for library construction must meet the following conditions: Total RNA quantity reaches the g level required for library construction, with a concentration of no less than 30 ng/L, an RQN value above 6.5, and an OD260/280 ratio between 1.8 and 2.2. Library construction uses the Illumina Stranded mRNA Prep, Ligation (Constant) Kit. Total RNA was extracted from 1 g of bone tissue samples from the control group and the 20 mg/L exposure group at day 28. The concentration and purity of the extracted RNA samples were determined using the Nano Drop 2000, and their integrity was assessed via agarose gel electrophoresis. The RNA quality was quantified using the Agilent 5300 platform (RQN). By using magnetic beads carrying oligothymidine (Oligo(dT)), which form A-T base complementary pairing with the polyA tail sequence at the 3 end of mRNA, mRNA molecules can be efficiently enriched from total RNA. To prepare the fragmented templates required for library construction, fragmenting buffer is added to randomly shear mRNA into small fragments of approximately 300 bp. Under the action of reverse transcriptase, using random primers, single-stranded cDNA is reverse transcribed from mRNA as a template, and further synthesized into double strands to form a stable double-stranded structure. The initial ends of the double-stranded cDNA are sticky ends, which must be converted to blunt ends using an end repair mix. A single adenine (A) base is then added to the 3 end to facilitate efficient connection with the adapter sequence. The adapter-linked products are purified and fragmented, and the selected fragments are amplified by PCR to obtain the final library. The experiment was sequenced on the NovaSeq X Plus platform, quantified using Qubit 4.0, and mixed according to the data ratio before sequencing. Bridge PCR amplification was performed on cBot to generate clusters. After obtaining the read counts of the genes sequenced in this project through quantitative analysis, DESeq2, DEGseq, or edgeR to analyze gene expression differences between the zebrafish control group and the 20 mg/L group, identifying differentially expressed genes. These genes were annotated using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database and the Gene Ontology (GO) database.