GSM7885423: RNASeq_H1Xsh_Untreated_r3; Homo sapiens; ncRNA-Seq
Total RNA of T47D-MTVL H1Xsh Untreated or 6-days-Dox-treated cells was extracted with High Pure RNA Isolation kit (Roche) Strand-Specific Transcriptome Library Construction Protocol (DNBSEQ). According to the manufacturer's instructions, the first step involves the removal of ribosomal RNA (rRNA) using target-specific oligos and RNase H reagents to deplete both cytoplasmic (5S rRNA, 5.8S rRNA, 18S rRNA, and 28S rRNA) and mitochondrial ribosomal RNA (12S rRNA and 16S rRNA) from total RNA preparations. Following SPRI beads purification, the RNA is fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments are copied into first-strand cDNA using reverse transcriptase and random primers, followed by second-strand cDNA synthesis using DNA Polymerase I and RNase H. This process removes the RNA template and synthesizes a replacement strand, incorporating dUTP in place of dTTP to generate ds cDNA. These cDNA fragments then have the addition of a single 'A' base and subsequent ligation of the adapter. After UDG treatment, the incorporation of dUTP quenches the second strand during amplification. The products are enriched with PCR to create the final cDNA library. The libraries' quality and quantity were assessed in two methods: check the distribution of the fragment size using the Agilent 2100 bioanalyzer and quantify the library using real-time quantitative PCR (QPCR) (TaqMan Probe). The qualified libraries were sequenced pair end on the BGISEQ-500/ MGISEQ-2000 System (BGI-Shenzhen, China).