GSM8454432: Itga9-Lyz2-cHet control, scRNAseq; Mus musculus; RNA-Seq
Single-cell RNA-seq experiment was performed by experimental personnel in the laboratory of NovelBio Co.,Ltd. The tissues were surgically removed and kept in MACS Tissue Storage Solution (Miltenyi Biotec) until processing. The 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 0.5mg/mL collagenase I, 0.5mg/mL collagenase II, 1mg/mL dispase II and 50 U/mL DNase I for 20 min at 37°C, with agitation. After digestion, samples were sieved through a 70µm cell strainer, and centrifuged at 400g for 5 min. After washing with PBS containing 0.04% BSA, the cell pellets were re-suspended in PBS containing 0.04% BSA and re-filtered through a 35μm cell strainer. 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 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 DNBSEQ-T7 Sequencer (MGI, Shenzhen, China) on a 150 bp paired-end run.