characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2021-10-03T00:15:50Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2021-10-03T00:15:50Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA10162940
characteristics.characteristic: External Id
characteristics.text: EMBL HEIDELBERG
characteristics.characteristic: INSDC center name
characteristics.text: 2021-10-03T00:15:50Z
characteristics.characteristic: INSDC first public
characteristics.text: 2021-10-03T00:15:50Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS7819377
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-10979:Sample 4
characteristics.characteristic: Submitter Id
characteristics.text: 8
characteristics.unit: week
characteristics.characteristic: age
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: primary cell line
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0003064
characteristics.characteristic: cell line
characteristics.text: mammary epithelial cell
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/CL_0002327
characteristics.characteristic: cell type
characteristics.text: house mouse
characteristics.characteristic: common name
characteristics.text: Protocols: The cells for the DNA extraction were harvested from three 3D gels per experimental condition by the digestion of the matrigel with collagenase and liberase for 1.5h at 37oC. Subsequently, the individually digested gels were pooled, washed three times with PBS and STOP medium (Lonza/Amaxa DMEM/F12 1:1 Mixture with HEPES, L-Gln, BE12-719F supplemented with HEPES to the final concentration of 25 mM and 10% Tet System Approved Fetal Bovine Serum, Biowest, S181T) and centrifuged shortly (1000 rpm, 2 min, at room temperature). Primary mammary epithelial cells were obtained through a digestion of mouse mammary glands in 5 mL of digestion medium (Lonza/Amaxa DMEM/F12 1:1 Mixture with HEPES, L-Gln, BE12-719F) supplemented with HEPES to the final concentration of 25 mM, 150 U/mL Collagenase type 3 (Worthington, LS004183), 20 μg/mL Liberase Blendzyme 2 (Roche, 05401020001) and 5 mL of Penicillin/Streptomycin (Gibco Life Technologies, 15140-122). The digestion for 15-16 hours at 37oC in 5% (vol/vol) CO2 atmopshere in loosely capped 50 mL polypropylene conical tubes was followed by a washing step with 45 mL of phosphate-buffered saline (PBS). Upon centrifugation at room temperature, 1000 rpm for 5 min, the interphase between the upper fat layer and the cell pellet was removed and 5 mL of 0.25% trypsin-EDTA (Invitrogen, 25200-056) was added. The suspension was incubated for 40 min at 37oC, 5% CO2 in loosely capped tubes, followed by a wash with 25 mL of STOP medium (Lonza/Amaxa DMEM/F12 1:1 Mixture with HEPES, L-Gln, BE12-719F supplemented with HEPES to the final concentration of 25 mM and 10% Tet System Approved Fetal Bovine Serum, Biowest, S181T) and a treatment with 5-15 mg/mL DNase I (ThermoFisher, 18068015). After another centrifugation step at room temperature, 1000 rpm for 5 min, the dissociated cells were resuspended in MEBM medium (Lonza, Mammary Epithelial Cell Basal Medium CC-3151 with supplements from Mammary Epithelial Cell Medium BulletKit CC- 3150) and plated onto collagen-coated plates (BD Biosciences, 356400) for the selection of epithelial cells. On the next day, the cells were washed with PBS and the remaining cells were treated with 500 μL of 0.25 % trypsin-EDTA until detachment. Trypsin was inactivated with 9 mL of STOP medium (described above), followed by a centrifugation step at room temperature with 1000 rpm for 5 min to obtain the primary mammary epithelial cells for seeding. To establish the 3D organoid culture the cell pellets were resuspended in PBS, counted, and mixed rapidly on ice with the prepared Matrigel-collagen mixture - Cultrex 3D Culture Matrix Basement Membrane Extract (Biozol, TRE-3445-005-01) and 1.5 mg/mL Cultrex 3D Collagen I rat tail (TEMA Ricerca, 3447-020-01). The mixed droplets in a volume of 100 μL, containing 12000 primary mouse mammary epithelial cells, were dispensed into flat bottom wells (Corning CellBIND 12 Well Clear Multiple Well Plates, 3336). After solidifying for 40 min at 37o C, 1.5 ml of MEBM serum-free medium (500 mL supplemented with 2 mL of bovine pituitary extract, 0.5 mL of hEGF, 0.5 mL of hydrocortisone, 0.5 mL of GA-1000, 0.5 mL Insulin from Mammary Epithelial Cell Medium BulletKit CC-3150) was added to each well and changed every second day. Subsequently, the cells were grown in the wells for 7 days until the 3D organoid culture had established including the formation of acini. After 7 days of 3D culture establishment the experiment was started by the addition of doxycycline (200 ng/mL) to the cell culture medium initiating transcription of the oncogenes and rapid cell proliferation. Tumor samples were harvested 5 days after oncogene induction. Subsequently, doxycycline was removed from the cell culture medium, which resulted in a silencing of the oncogenes followed by tumor regression. Residual samples were harvested 7 days after de-induction (12 days in total). Non-induced structures were grown in medium without the addition of doxycycline and sampled in parallel at the same time point as the residual cells (day 12). The medium was changed every second day with the exception of the doxycycline-induced cultures at the days three to eight, during which it was changed daily. The DNA extraction from the harvested cell pellet was done following the Qiagen protocol for cultured cells (Qiagen Blood & Cell Culture DNA Mini kit, 13323). The library for Enzymatic Methyl-seq (EM-seq) was prepared with the NEBNext enzymatic Methyl-seq Kit (NEW ENGLAND BioLabs Inc.) from 100 ng of sheared DNA. According to the protocol’s recommendations 2 ng of lambda phage DNA and 0.1 ng of pUC19 plasmid DNA were spiked-in per 100 ng sample DNA prior to fragmentation. The fragmentation of the DNA mix was done using a Covaris S2 focused-ultrasonicator.
characteristics.characteristic: description
characteristics.text: adult
characteristics.characteristic: developmental stage
characteristics.text: food pellets TD (Envigo)
characteristics.characteristic: diet
characteristics.text: tumor
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0000616
characteristics.characteristic: disease
characteristics.text: TetO-cMYC/TetO-Neu/MMTV-rtTA
characteristics.characteristic: genotype
characteristics.text: Lonza MEBM serum free media (CC-3151), 500 mL supplemented with 2 mL of bovine pituitary extract, 0.5 mL of hEGF, 0.5 mL of hydrocortisone, 0.5 mL of GA-1000 and 0.5 mL Insulin from Mammary Epithelial Cell Medium BulletKit (CC-3150)
characteristics.characteristic: growth condition
characteristics.text: Mouse 503
characteristics.characteristic: individual
characteristics.text: Mus musculus
characteristics.characteristic: organism
characteristics.text: Mus musculus
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_10090
characteristics.characteristic: organism
characteristics.text: mammary gland
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/BTO_0000817
characteristics.characteristic: organism part
characteristics.text: Mus musculus
characteristics.characteristic: scientific_name
characteristics.text: female
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/PATO_0000383
characteristics.characteristic: sex
characteristics.text: FVB
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0007728
characteristics.characteristic: strain
characteristics.text: Sample 4
characteristics.characteristic: title