title: Contractile fibroblasts form a transient niche for the branching mammary epithelium (fibroblasts)
library_construction_protocol: Experimental animals were obtained by breeding of the parental strains, the genotypes were determined by genotyping. The mice were housed in individually ventilated or open cages, all with ambient temperature of 22°C, a 12 h:12 h light:dark cycle, and food and water ad libitum. Microdissected pieces of mammary gland pooled from 11 animals were digested in a collagenase/hyaluronidase solution [3 mg/ml collagenase A, 100 U/ml hyaluronidase (all Merck), 5% fetal bovine serum (FBS; Hyclone/GE Healthcare) in CO2 independent medium (Thermo Fisher Scientific)] for 2 h at 37°C, shaking at 120 rpm. Resulting tissue fragments were treated with 0.25% trypsin, 5 mg/ml dispase II, 100 µg/ml DNase and red blood cell lysis buffer (Sigma) and filtered through a 40 µm cell strainer. Resulting single cell suspension was spun down and resuspended in staining medium (10% FBS, 100 U/ml of penicillin, and 100 μg/ml of streptomycin in CO2 independent medium) with antibodies for negative selection (CD45-APC/Cy7 from BD Biosciences and CD31-APC/Cy7, CD24-APC/Cy7, CD49f-APC/Cy7 from BioLegend) and incubated on ice in dark for 30 min. Then, the cells were washed twice with PBS, resuspended in flow buffer (5 mM EDTA, 1% BSA, 1% FBS in phenol red-free DMEM/F12 with HEPES) and filtered through a 40 µm cell strainer. The analysis was done on  Fusion (BD), operated by SORP software (BD). The resulting cell pellet was pooled down, resuspended in freezing medium (10% DMSO in FBS) and frozen by slow freezing at -80°C and moved to liquid nitrogen 24 h thereafter. For microdissection of TEB- and duct-containing regions, 5 weeks old R26-mTmG females were used. Mammary glands #3 and #4 were dissected and spread on 10 cm plastic petri dishes. Under the control of a fluorescent stereoscope (Leica FM165C), 0.5×0.5 mm pieces of tissue containing either TEBs or pieces of ducts (assessed based on tdTomato fluorescence) were dissected and collected in CO2 independent medium on ice. 5-8 mice were pooled for one experiment. Single-cell suspensions were prepared and processed using the GEM-X Single Cell 3' v4 kit (10x Genomics) following the manufacturer's protocol for frozen cells. Nucleic acid libraries were prepared and processed using the GEM-X Single Cell 3' v4 kit (10x Genomics) following the manufacturer's protocol for frozen cells.