GSM9325559: CD45+ immune cells, control or telmisartan-treated [Combined_WTA]; Mus musculus; RNA-Seq
Subcutaneous tumor tissues were harvested and mechanically dissociated into small pieces. The tissue was digested using a mixture of Collagenase II/IV, DNase I, and Hyaluronidase in RPMI-1640 medium at 37°C for 60 min with agitation. The digestion was stopped with serum-containing medium, and the suspension was filtered through a 70-μm cell strainer. Red blood cells were removed using a lysis buffer. The resulting single-cell suspension was washed with PBS and stained with a viability dye and anti-CD45 antibodies. Live CD45+ immune cells were isolated by Fluorescence-activated cell sorting (FACS). To distinguish between the drug-treated and control groups, sorted cells from each group were labelled with distinct BD Mouse Immune Single-Cell Multiplexing Sample Tags according to the manufacturer's instructions. After washing, the labelled cells were pooled together for subsequent capture. Single-cell libraries were constructed using the BD Rhapsody™ Single-Cell Analysis System (BD Biosciences) following the manufacturer's instructions. Briefly, the pooled single-cell suspension was loaded into a BD Rhapsody™ Cartridge to capture single cells with Cell Capture Beads in microwells. Cells were lysed, and poly-A mRNA, along with Sample Tag oligos, were captured onto the beads via hybridization. The beads were then retrieved magnetically, and cDNA was synthesized via reverse transcription. Following Exonuclease I treatment to remove unbound oligos, the library preparation was divided into two workflows: (1) For Whole Transcriptome Analysis (WTA), the cDNA underwent Random Priming and Extension (RPE) followed by PCR amplification; (2) For Sample Tag libraries, the tagged sequences were specifically amplified using Sample Tag PCR primers. Both libraries were indexed using Illumina-compatible P5 and P7 sequences, purified using AMPure XP magnetic beads, and quantified using a Qubit Fluorometer prior to sequencing.