GSM9632589: Human ovarian cancer tissue 3; Homo sapiens; ncRNA-Seq
After grinding the tissue in liquid nitrogen, a specific lysis buffer was added. Following centrifugation, 200 μL of the lysate was collected, and total RNA was extracted using the Takara RNAiso Plus method. The extracted total RNA was then subjected to quality control. Subsequently, the tissue lysate was applied to MicroSpin S-400 HR Columns to separate ribosomal complexes. RNA was then extracted from the isolated ribosomal complexes using the Takara RNAiso Plus method. After the samples passed quality control, libraries were constructed using the Total RNA-seq (H/M/R) Library Prep Kit for Illumina®. First, designed DNA probes were hybridized with the RNA samples to remove rRNA from total RNA. Subsequently, the RNA was fragmented, and first-strand cDNA was synthesized. Using a strand-specific approach, dUTP was incorporated during second-strand cDNA synthesis to label the second strand, while end repair was completed in the same step. This was followed by A-tailing, adapter ligation, purification of the ligation products, size selection, and library amplification. Prior to PCR amplification, the dUTP-containing second-strand cDNA template was digested using UDG enzyme. After amplification, the libraries were purified and recovered using magnetic beads, yielding the final RNA-seq libraries.