Single cell RNA sequencing of dormant and proliferative PyMT-Bo1 breast cancer cells isolated from murine bone

Transcriptome profiling of dormant (DiD+) and proliferative (DiD-) PyMT-Bo1 isolated by FACS from bone 11 days after intracardiac (IC) injection. PyMT-Bo1 cells were first labelled with membrane DiD dye before injected into mice. Femur and tibia were isolated and pooled together from 5 IC injected mice. 32 dormant (DiD+) and 28 proliferative (DiD-) cells were isolated by FACS sorting for single cell RNA sequencing (scRNA-seq).Single‐cell cDNA was generated using the SMARTer Ultra Low RNA Kit v4 (Takara) with modifications. 1:2,500,000 dilution of ERCC spike-in controls (Ambion) were incorporated during first-strand cDNA synthesis and subsequent steps were performed according to the manufacturer's instructions at half-reaction volumes. cDNA amplification was performed at 18 cycles and its quality was assessed using the Bioanalyzer HS DNA chip (Agilent Technologies) according to the manufacturer's instructions. Sequencing libraries were generated using 1 ng of input material using the Nextera XT Kit (Illumina) according to the manufacturer's protocol at half-reaction volumes. Libraries were pooled and paired-end sequenced (150-bp reads) on Illumina NextSeq500 on a high-throughput mode. Illumina sequence adapters were trimmed and reads were aligned to a modified version of the GRCm38/mm10 mouse genome (supplemented with ERCC, mApple, eGFP, luciferase, PyMT oncogene sequences) using the STAR aligner. Summarized gene transcript counts and TPMs were generated using RSEM.