RNA-Seq of 4T1 primary tumor-4
The purified RNA was used for the preparation of the sequencing library by TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, CA, USA) following the manufacturers recommendations. Briefly, mRNA was purified from total RNA (1 g) by oligo(dT)-coupled magnetic beads and fragmented into small pieces under elevated temperature. The first-strand cDNA was synthesized using reverse transcriptase and random primers. After the generation of double-strand cDNA and adenylation on 3 ends of DNA fragments, the adaptors were ligated. The products were enriched with PCR and purified with AMPure XP system (Beckman Coulter, Beverly, USA). The libraries were qualified by Qsep400 System (Bioptic Inc., Taiwan) and quantified by Qubit 2.0 Fluorometer (Thermo Scientific, Waltham, MA, USA). The qualified libraries were then sequenced on an Illumina NovaSeq 6000 platform with 150 bp paired-end reads generated by Genomics, BioSci & Tech Co., New Taipei City, Taiwan.