GSM8966573: 786--O cell,Si-TRIM28-1; Homo sapiens; RNA-Seq
Total RNA was extracted from Parental 786-O renal carcinoma cells, TRIM28 knockdown cells, and PARP1 knockdown cells using the  TRIzol Reagent (Invitrogen)] according to the manufacturer's instructions. Briefly, cells were lysed directly in culture dishes with TRIzol reagent, followed by phase separation with chloroform. The aqueous phase was collected, and RNA was precipitated with isopropanol, washed with 75% ethanol, and dissolved in RNase-free water. RNA concentration and purity were assessed using a NanoDrop spectrophotometer, and RNA integrity was confirmed by agarose gel electrophoresis. mRNA-seq libraries were prepared using the  NEBNext Ultra II Directional RNA Library Prep Kit for Illumina  following the manufacturer's protocol. Briefly, poly(A)+ RNA was enriched from total RNA using oligo(dT) magnetic beads. The mRNA was then fragmented and reverse-transcribed into first-strand cDNA using random primers and reverse transcriptase. Second-strand cDNA synthesis was performed to generate double-stranded cDNA. End repair, A-tailing, and adapter ligation were carried out, followed by PCR amplification to enrich the libraries. The final libraries were purified and quality-checked using Agilent Bioanalyzer 2100. Libraries were then quantified and pooled for sequencing on an Illumina NovaSeq 6000 platform.