Single cell RNA analysis of breast cancer bone metastases

For single cell RNA sequencing analysis, whole cell population isolated from two breast cancer bone metastases and associated organoid models were analyzed by 10X genomic single cell RNA platform. Raw BCL files from Novaseq6000 was converted to FASTQ files with cellranger (Version: 3.1.0) mkfastq function. Cellranger count function was then applied for read alignment, barcode and UMI counting using GRCh38 as reference. scCB2 and DoubletFinder were used to predict and remove empty droplets and doublets of single cell data. Cells with number of features less 300 and more than 9000 were further filtered out based on the distribution of number of features. For bulk RNA sequencing, RNA were extracted from FFPE preserved primary tumor and bone metastasis. Library was prepared with TruSeq Stranded mRNA for FFPE kit, and sequenced by NextSeq 500 with high output 150 cycle kit. Overall design: Single cell and bulk RNA sequencing of two synchronous bilateral breast cancer bone metastases with associated patient derived organoid models and paired primary tumor.