title: GSM7687947: Male, CD45 negative cells, scRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: The MB49 tissue samples were processed as described below. Briefly, samples were first washed with phosphate-buffered saline (PBS), minced into small pieces (approximately 1mm3) on ice and enzymatically digested with 10 U/mL collagenase I (Worthington) and 400 U/mL collagenase Ⅳ (Worthington) and 30 U/mL DNase I (Worthington) and 1mg/mL Dispase Ⅱ (Worthington) for 45 min at 37°C, with agitation. After digestion, samples were sieved through a 70µm cell strainer, and centrifuged at 300 g for 5 min. After the supernatant was removed, the pelleted cells were suspended in red blood cell lysis buffer (Miltenyi Biotec) to lyse red blood cells. After washing with PBS containing 0.04% BSA, the cell pellets were stained with mouse CD45 antibody and underwent flow sorting to obtain live CD45- and CD45+ cells. Dissociated single cells were then stained for viability assessment using Calcein-AM (Thermo Fisher Scientific) and Draq7 (BD Biosciences). BD Rhapsody system was used to capture the transcriptomic information of the male and female mouse MB49 tumor derived CD45+ and CD45- 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.