title: GSM6433243: P0, WT, scRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: For mammary rudiments at E13.5, E14.5 and E15.5, single cell dissociation was performed through enzymatic digestion with 300 U/ml collagenase A and 300 U/ml hyaluronidase for 90 minutes at 37°C under shaking. Cells were further treated with 0.1 mg/ml DNase I for 3 minutes. 10% FBS diluted in PBS was added to quench the DNase I. For mammary glands at P0, single cell dissociation was performed through enzymatic digestion with 600 U/ml collagenase A and 150 U/ml hyaluronidase for 90 minutes at 37°C under shaking were used for enzymatic digestion. Cells were further treated with 0.1 mg/ml DNase I for 3 min and an additional incubation in 0.63% NH4Cl for 1 min allowed lysis of red blood cells. Cells were pelleted by centrifugation at 320 g for 10 minutes. For all developmental times, after careful removal of the supernatant, cells were incubated in fluorescently labelled primary antibodies for 15 minutes on ice. Cells were washed from unbound antibodies with 2% FBS in HBSS and the cell suspension was passed through a 40 µm cell strainer filter to eliminate cell clumps. Cell viability was determined with DAPI and doublets were systematically excluded during analysis. CD45+, CD31+, Ter119+ (Lin+) non-epithelial cells were excluded. FACS analysis was performed using an ARIA flow cytometer (BD). Single cell capture and library construction were performed using the 10x Genomics Chromium Single Cell 3' v3.1 kit following the manufacturer's instructions. Briefly, sorted mammary gland cells were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, P5 and P7 primers used for Illumina bridge amplification and a sample index were added.