TB 1 (CRX1524859)
The femurs were harvested and digested from both HCC tumor-free and tumor-bearing mice, followed by sorting live cells utilizing Fluorescence-activated Cell Sorting (FACS) for subsequent scRNA-seq analysis. ScRNA-seq was performed with help from NovelBio Bio-Pharm Technology Co., Ltd. The dissociated single cells were stained with AO/PI for viability assessment using Countstar Fluorescence Cell Analyzer.  BD Rhapsody system was used to capture the transcriptomic information of the single cells. Single-cell capture was achieved by random distribution of a single-cell suspension across>200,000 microwells through a limited dilution approach. Beads with oligonucleotide barcodes were added to saturation so that a bead was paired with a cell in a microwell. The cells were lysed in the microwell to hybridize mRNA molecules to barcoded capture oligos on the beads. Beads were collected into a single tube for reverse transcription and ExoI digestion. Upon cDNA synthesis, each cDNA molecule was tagged on the 5' end (that is, the 3' end of a mRNA transcript) with a unique molecular identifier (UMI) and cell barcode indicating its cell of origin. Whole transcriptome libraries were prepared using the BD Rhapsody single-cell whole-transcriptome amplification (WTA) workflow including random priming and extension (RPE), RPE amplification PCR and WTA index PCR. The libraries were quantified using a High Sensitivity DNA chip (Agilent) on a Bioanalyzer 2200 and the Qubit High Sensitivity DNA assay (Thermo Fisher Scientific). Sequencing was performed by illumina sequencer (Illumina, San Diego, CA) on a 150 bp paired-end run.