Next Generation Sequencing Facilitates Quantitative Analysis of ALDH+ E-BCSC, CD24-CD44+ M-BCSC and Bulk tumor cell Transcriptomes from MC1 and Vari068 PDX models of TNBC

We report the application of single-molecule-based sequencing technology for high-throughput profiling of genes in E-, M-BCSCs and bulk tumor cells in two PDX models of triple negative breast cancer . Examination of global gene expression in E and M-BCSCs and Bulk tumor cells in 2 PDXs. Total RNA was extracted from E- (H2Kd-ALDH+), M- (H2Kd-CD44+/CD24-) BCSC-enriched and bulk (H2Kd-ALDH-CD44-/CD24+) cell populations freshly sorted from dissociated tumor cells of Vari068 and MC1 PDXs. Total RNA was extracted with RNeasy Mini Kit (Qiagen) according to manufacturer's instructions, and 500 ng was then taken out from each of them and subjected to mRNA isolation using NEBNext Poly (A) mRNA Magnetic Isolation Module (NEB, Ipswich, MA, USA). The mRNA library was constructed using NEBNext UltraTM RNA Library Prep Kit for Illumina (NEB, Ipswich, MA) according to the instruction manual, which includes sequential RNA fragmentation, reverse transcription using random primers, second strand cDNA synthesis, end repair, dA-tailing, adapter ligation, U excision, and PCR enrichment. mRNA libraries were sequenced on an Illumina HiSeq X Ten platform with 150bp pair-end reads. All reads passed filter were trimmed to remove low-quality bases and adaptor sequences. Reads were then aligned to the mm10 reference genome using tophat2 (v2.0.13), and FPKMs were calculated.