GSM6257309: BMBAs and basophils under homeostatic conditions (hashtag library); Mus musculus; RNA-Seq
For scRNA-seq of bone marrow-derived basophils (BMBAs), bone marrow cells cultured in the presence of IL-3 (0.3 ng/mL: BioLegend) for 3 or 7 days were subjected to scRNA-seq analysis. For scRNA-seq of basophils under homeostatic conditions, single cell samples were prepared from the bone marrow and spleen of Mcpt8GFP transgenic mice and depleted of lineage-positive cells by using biotinylated lineage antibodies (anti-CD4, anti-CD8a, anti-CD19, anti-B220, anti-Gr-1 and anti-TER-119) and EasySep Mouse Streptavidin RapidSpheres Isolation Kit (Stemcell Technologies). GFP-positive cells were sort-purified from lineage-negative cells and subjected to scRNA-seq analysis Library construction was conducted according to the TAS-seq protocol reported in previous report (Shichino et al. bioRxiv 2022). Briefly, cells were indexed with Totalseq anti-mouse Hashtag antibody (BioLegend) for multiplexing. Reverse-transcription was conducted by using the BD Rhapsody system according to the manufacturer's instruction. Preparation of cDNA library and hashtag library was performed as described previously (Chen CY et al. Cancer Immunol Res 2021). Sequencing libraries were prepared by utilizing NEBNext UltraII FS Library Prep Kit (New England BioLabs, USA).