RNA-Seq of Homo sapiens: adult female endometrium
Total RNA was extracted from paired endometrial cancer and adjacent normal tissues using TRIzol reagent (Invitrogen) and purified with the RNeasy Mini Kit (Qiagen). RNA integrity was confirmed via Agilent Bioanalyzer 2100 (RIN 7.0). Poly(A)+ mRNA was isolated using oligo(dT) magnetic beads. mRNA was fragmented to ~200 bp via heat treatment (94C, 5 min) and reverse-transcribed into double-stranded cDNA using SuperScript II (Invitrogen) and random hexamers. cDNA ends were repaired, A-tailed, and ligated with DNBSEQ-specific indexed adapters (DNBSEQ RNA Library Prep Kit, BGI). Libraries were amplified with 12 cycles of PCR using Phusion DNA polymerase (NEB), then purified and validated for size distribution on the Agilent Bioanalyzer. Quantification was performed with Qubit dsDNA HS Assay Kit. Sequencing was conducted on the DNBSEQ-T7 platform using DNA nanoball (DNB) technology with 100 bp paired-end reads.