Two phenol-chloroform methods were used to purify RNA. First, isolate the total RNA from the specimens by employing suitable methodologies, such as the phenol-chloroform extraction technique, which exploits the differential solubility of RNA in organic solvents, or column-based purification systems that rely on the specific binding properties of RNA to stationary phases. Subsequently, conduct a rigorous quality control assessment on the RNA samples to guarantee their structural integrity and high purity. This can be accomplished by visualizing the 28S and 18S ribosomal RNA (rRNA) bands on an electropherogram or agarose gel, where the intensity and distinctness of these bands serve as indicators of RNA integrity. Additionally, measure the RNA Integrity Number (RIN) using a bioanalyzer, which provides a quantitative evaluation of RNA quality based on its fragmentation pattern. In the case of whole-transcriptome sequencing, it is imperative to deplete ribosomal RNA (rRNA) to selectively enrich the transcripts of biological interest. For messenger RNA sequencing (mRNA-seq), specifically target and enrich polyadenylated (poly-A) transcripts, which are characteristic of mature mRNAs. Following these enrichment steps, convert the RNA into complementary DNA (cDNA) through the process of reverse transcription, utilizing reverse transcriptase enzymes that catalyze the synthesis of DNA from an RNA template. Subsequently, ligate adapters to the cDNA fragments. These adapters play a crucial role in the subsequent sequencing process, providing essential binding sites for the sequencing platform and enabling the proper orientation and attachment of the cDNA molecules. Finally, carry out polymerase chain reaction (PCR) amplification to exponentially increase the quantity of DNA, thereby constructing the cDNA library. Once the library is successfully constructed, it is primed and ready for sequencing on the appropriate next-generation sequencing platform, which will generate the necessary data for downstream transcriptomic analysis.