title: GSM4761644: Cell_line_3_off_dox; Mus musculus; RNA-Seq
library_construction_protocol: Total RNA was extracted from fresh or flash-frozen mammary tumors or cultured cancer cells using the RNeasy Mini Kit (QIAGEN). The protocol, provided by QIAGEN, was followed to extract the total RNA. The concentration of the RNA was determined on a NanoDrop spectrophotometer, and the integrity of the RNA was validated using gel electrophoresis. Quality and quantity of the total RNA was re-confirmed by Novogene. Only high quality RNA was used for library prepration. Libreary construction was performed by Novogene (After the QC procedures, mRNA is enriched using oligo(dT) beads. First, the mRNA was fragmented randomly by adding fragmentation buffer, then the cDNA was synthesized by using mRNA template and random hexamers primer, after which a custom second-strand synthesis buffer (Illumina) , dNTPs, RNase H and DNA polymerase I were added to initiate the second-strand synthesis. Second, after a series of terminal repair, A ligation and sequencing adaptor ligation, the double-stranded cDNA library was completed through size selection and PCR enrichment. The quality of the library was confirmed via these three steps: (1) Qubit 2.0: tests the library concentration preliminarily. (2) Agilent 2100: tests the insert size. (3) Q-PCR: quantifies the library effective concentration precisely.