title: duct
organism: Mus musculus
description: Protocols: 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 8 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 flow buffer (5 mM EDTA, 1% BSA, 1% FBS in phenol red-free DMEM/F12 with HEPES) for single cell RNA sequencing 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- or 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.
attributes.tag: ENA first public
attributes.value: 2025-08-07
attributes.tag: INSDC center name
attributes.value: Institute of Molecular Genetics of the Czech Academy of Sciences
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-15259:duct
attributes.tag: age
attributes.value: 5
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell type
attributes.value: all cells
attributes.tag: collection date
attributes.value: not collected
attributes.tag: common name
attributes.value: house mouse
attributes.tag: developmental stage
attributes.value: puberty
attributes.tag: genotype
attributes.value: R26-mTmG homozygous
attributes.tag: geographic location (country and/or sea)
attributes.value: not collected
attributes.tag: individual
attributes.value: NA
attributes.tag: isolate
attributes.value: not applicable
attributes.tag: organism part
attributes.value: mammary gland duct region
attributes.tag: sample name
attributes.value: E-MTAB-15259:duct
attributes.tag: scientific_name
attributes.value: Mus musculus
attributes.tag: sex
attributes.value: female
attributes.tag: strain
attributes.value: R26-mTmG