GSM9156331: sgKMT5A1; Homo sapiens; RNA-Seq
Total RNA was extracted using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. The quality and concentration of RNA were assessed using a NanoDrop spectrophotometer and an Agilent 2100 Bioanalyzer to ensure RNA integrity (RIN > 8.0) before library preparation. mRNA was isolated with Dynabeads Oligo(dT) (Thermo Fisher Scientific), fragmented using the Magnesium RNA Fragmentation Module (NEB). First-strand cDNA synthesis was performed using SuperScript™ II Reverse Transcriptase (Invitrogen). Second-strand cDNA synthesis was carried out using E. coli DNA Polymerase I (NEB), RNase H (NEB), and dUTP solution (Thermo Fisher Scientific). The double-stranded cDNA was end-repaired, A-tailed, and ligated with Illumina-compatible adapters using the NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs, USA). Adapter-ligated cDNA fragments were size-selected (300-500 bp) using AMPure XP beads (Beckman Coulter, USA) and subsequently amplified by PCR. The average insert size for the final cDNA libraries was 300 ± 50 bp.