GSM9093923: Mouse mammary gland, KO group (combined three mice); Mus musculus; RNA-Seq
Mammary glands from both groups were dissected and mechanically dissociated. The finely minced tissue was transferred to a digestion mix consisting of DMEM/F12 (Gibco) + 10 mM HEPES (Gibco) + collagenase (Roche) + 200 U ml⁻¹ hyaluronidase (Sigma) + gentamicin (Gibco) for 3 hours at 37°C and vortexed every 30 minutes. After the lysis of red blood cells in NH4Cl, cells were briefly digested with warm 0.05% Trypsin-EDTA (Gibco), 5 mg ml−1 dispase (Sigma), and 1 mg ml−1 DNase (Sigma), and filtered through a cell strainer (BD Biosciences) to remove debris, yielding a single-cell preparation for sequencing. Propidium iodide (PI, Sigma) was used at a working concentration of 1 μg/ml, prepared by diluting the stock solution 1:1000 to detect dead cells. After sorting, cells were spun down and resuspended. Samples were manually counted using an improved Neubauer chamber, and the cell concentration was normalized. Equal numbers of cells per sample were processed for scRNA library preparation. ScRNA-seq libraries for 5Ht and 6Ho mice were generated using the 10X Genomics Chromium platform.