2025-08-07
ENA first public
ERC000011
ENA-CHECKLIST
Institute of Molecular Genetics of the Czech Academy of Sciences
INSDC center name
public
INSDC status
ERS25081713
SRA accession
E-MTAB-15259:duct
Submitter Id
5
week
age
ArrayExpress
broker name
all cells
cell type
not collected
collection date
house mouse
common name
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.
description
puberty
developmental stage
R26-mTmG homozygous
genotype
not collected
geographic location (country and/or sea)
NA
individual
not applicable
isolate
Mus musculus
organism
Mus musculus
organism
mammary gland duct region
organism part
Mus musculus
scientific_name
female
sex
R26-mTmG
strain
duct
title