GSM8526452: triple negative breast cancer, OPG_Fc rep2; Mus musculus; RNA-Seq
Total RNA was extracted using the NZY Total RNA Isolation kit (#MB13402, Nzytech), according to manufacturer's instructions. Tumor tissues preserved in RNA later (#AM7021, Thermo Fisher Scientific) at necropsy were transferred to lysis buffer with silica beads (#110791102; Biospec Products), and disrupted in a Minibeadbeater (Biospec Products) for 1-2 min. mRNA enrichment was performed on total RNA using oligo(dT)-attached magnetic beads. The enriched mRNA with poly(A) tails was fragmented using a fragmentation buffer, followed by reverse transcription using random N6 primers to synthesize cDNA double strands. The synthesized double stranded DNA was then end-repaired and 5'-phosphorylated, with a protruding 'A' at the 3' end forming a blunt end, followed by ligation of a bubble-shaped adapter with a protruding 'T' at the 3' end. The ligation products were PCR amplified using specific primers. The PCR products were denatured to single strands, and then single-stranded circular DNA libraries were generated using a bridged primer. The constructed libraries were quality-checked and sequenced after passing the quality control.