GSM7881579: T3-T4 Normal_(LN negative patient 04) [RT2107138140-1]; Homo sapiens; RNA-Seq
The samples were collected from patients who underwent surgery at the Nanfang Hospital of Southern Medical University (Guangzhou, China) between December 2020 and December 2021. All samples were immediately snap-frozen in liquid nitrogen and stored at −80 °C until required. The extracted total RNA is subjected to quality checks for purity, quantity and integrity (RIN). Isolation: Because eukaryotic mature mRNA has a ploy(A) tail and other RNAs do not, oligo dT magnetic beads can be used to specifically bind to the poly(A) tail of mRNA to remove other RNAs. fragmentation: Fragmentation of purified mRNA with reagents (fragmentation reagent). cDNA first strand synthesis Random hexamer primer, reverse transcriptase to synthesise cDNA using mRNA as template. cDNA second strand synthesis: Second strand synthesis and deletion of mRNA to produce double-stranded cDNA (ds cDNA). Purification of double-stranded cDNA: end repair of double-stranded cDNA. Ends are patched and the repaired cDNA is then purified. ds cDNA 3' end is added with A (dA - tailing). The ligand: each ligand has an index (6bp) and different libraries can be built using different indexes. PCR enrichment of the libraries: After amplification and purification, the libraries will be checked and ready for sequencing.