Illumina HiSeq 4000 paired end sequencing; Time-resolved single-cell analysis of Brca1 associated mammary tumorigenesis reveals aberrant differentiation of luminal progenitors
Time-resolved single-cell analysis of Brca1 associated mammary tumorigenesis reveals aberrant differentiation of luminal progenitors
Lymph node divested mouse mammary glands (excluding the cervical pair) were mechanically dissociated after collection, pooled per animal and the finely minced tissue was transferred to DMEM/F12 (Gibco) + 10 mM HEPES (Gibco) + 2 mg ml-1 collagenase (Roche) + 200 U ml−1 hyaluronidase (Sigma) (CH) + gentamicin (Gibco) at 37 °C and vortexed every 30 min. 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).    Frozen vials of human epithelial-enriched fractions dissociated as previously described20 or of organoids from the Breast Cancer Now tissue bank were defrosted and diluted in cold HBSS 1% FCS (HF), further digested with warm Trypsin-EDTA (Gibco), 5 mg ml−1 dispase (Sigma) and 1 mg ml−1 DNase (Sigma) and filtered through a 40uM cell strainer (BD Biosciences).  MACS Dead Cell Removal kit was used to exclude dead cells from single cell suspensions. Subsequently, cells were spun down and resuspended in HF. Samples were manually counted using an improved Neubauer chamber and the cell concentration was normalised by addition of HF. Equal numbers of cells per sample were processed for scRNA library preparation. Nucleic acid extraciton was performed according to the 10x v2/v3 manual. Samples were processed for first strand cDNA synthesis within 6 h from tissue isolation. The remaining steps of library preparation were completed within the following 7 days. The library construction was performed according to the 10x v2/v3 manual.