GSM9825567: NC1_uterus; Mus musculus; RNA-Seq
Total RNA was extracted from mouse uterine tissue and primary uterine fibroblasts using TRIzol reagent (Invitrogen, USA) following the manufacturer's standard protocol. RNA integrity was assessed via agarose gel electrophoresis, and RNA purity and concentration were quantified using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). Only RNA samples with OD260/280 between 1.8–2.1, OD260/230 >1.8 and RNA integrity number (RIN) ≥7.0 were retained for subsequent library construction. Genomic DNA contamination was eliminated by DNase I digestion. mRNA was enriched from qualified total RNA using oligo(dT) magnetic beads to capture polyA-tailed transcripts. Fragmentation buffer was added to interrupt mRNA into short fragments of 200–300 bp. Using fragmented mRNA as template, random hexamer primers were applied to synthesize first-strand cDNA, followed by second-strand cDNA synthesis with DNA polymerase I. After end repair, A-tailing and adapter ligation, cDNA fragments were purified and size-selected. PCR enrichment was performed to construct strand-specific RNA-seq libraries. Library quality was verified by Agilent 2100 Bioanalyzer, and qualified libraries were sequenced on an Illumina NovaSeq 6000 platform with paired-end 150 bp (PE150) sequencing strategy.