GSM8775515: Mutiplexed samples; Mus musculus; RNA-Seq
Brains collected after intracardiac perfusion with 20ml of PBS were dissociated into single-cell suspensions using the mouse tumor dissociation kit (#130-096-730) and the gentleMACS Octo Dissociator according to the manufacturer's protocol (Miltenyi Biotec). Single-cell libraries of dissociated single cells were prepared using the BD RhapsodyTM platform (BD Biosciences). Single-cell suspensions obtained from the brains of individual tumor-bearing mice were stained with oligonucleotide-conjugated Sample Tags using the mouse Single-Cell Multiplexing Kit (cat# 633793) following the manufacturer's protocol (BD Biosciences). Sorted live cells from barcoded samples were counted and pooled at equal ratios and loaded onto a BD Rhapsody cartridge for capture. Cell capture and library preparation were completed using the BD Rhapsody Whole Transcriptome Analysis (WTA) Amplification Kit (cat# 633801). Briefly, cells were captured with beads in microwell plate, followed by cell lysis, bead retrieval, cDNA synthesis, template switching and Klenow extension, and library preparation following the BD Rhapsody protocol. The quality of the final libraries was assessed by using an Agilent's Bioanalyzer high sensitivity analysis and concentrations were measured by a Qubit method. The final libraries were diluted and normalized to 4nM and then pooled for paired-end sequencing on NovaSeq 6000 sequencer (Illuminia, San Diego, CA) in Novogene (Novogene Corporation Inc., Sacramento, CA).