Illumina HiSeq 2500 sequencing; Detection and removal of barcode swapping in single-cell RNA-seq data
Detection and removal of barcode swapping in single-cell RNA-seq data
Tissues were harvested at gestation day 14.5 (G), lactation day 6 (L) and day 11 post natural weaning of the pups (PI). Tissue from NP females was harvested at 8 weeks of age. Two individual mice per developmental time point were used in the study. Lymph node divested mammary glands (excluding the cervical pair) were dissected from the mice 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) and incubated for 3 hours at 37° C.  Next, single cell suspensions were incubated in HF medium (Hank's balanced salt solution (Gibco) + 1% foetal bovine serum, Gibco) + 10% normal rat serum (Sigma) for 20 min on ice to pre-block before antibody staining. All antibody incubations were performed for 10 min on ice in HF media. Mammary cells were stained with the following primary antibodies: CD31-biotin (eBioscience, clone 390); CD45-biotin (eBioscience, clone 30F11); Ter119-biotin (eBioscience, clone Ter119) and EpCAM-PE (Biolegend, clone G8.8). Cells were then stained with streptavidin-PE-CF594 (BD-Biosciences). Propidium Iodide (PI, ; Sigma) was used to detect dead cells. Cells were filtered through a cell strainer (Partec) before sorting. Sorting of cells was done using a SH800Z sorter (SONY). Single-stained control cells were used to perform compensation manually and unstained cells were used to set gates. Chip alignment and sorting calibration was performed with automatic setup beads (SONY) immediately prior to sorting. Doublets, dead cells and contaminating haematopoietic, endothelial and stromal cells were gated out and EpCAM positive cells were sorted in LoBind microcentrifuges tubes (Eppendorf) with HF. After sorting cells were spun down and resuspended in of HF. Samples were manually counted using an improved Neubauer chamber and the cell concentration was normalized by addition of HF. Equal numbers of cells per sample were processed for scRNA library preparation. Samples were processed for scRNA library preparation within 9 hours from tissue isolation. For droplet-based scRNA-seq, sorted cells were encapsulated into droplets, and libraries were prepared using the ChromiumTM Single Cell 3' Reagent Kits v2 according to manufacturer's protocol (10x Genomics). For droplet-based scRNA-seq, sorted cells were encapsulated into droplets, and libraries were prepared using the ChromiumTM Single Cell 3' Reagent Kits v2 according to manufacturer's protocol (10x Genomics).
Experimental Factor: sampling time point
post-involution
Experimental Factor: protocol
HiSeq2500