biostudies-arrayexpressYura SongMus musculushttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-16111To mimick menopause hormone therapy and compare the effects of estetrol (E4) to the ones of estradiol (E2), ovariectomized mice were treated for 2 weeks with E2, a physiological dose of E4 (E4low) or a supra physiological dose of E4 (E4hi). Non treated ovariectomized mice were used as control. Basal cells, estrogen positive luminal cells and estrogen negative luminal cells from MG were sorted based on surface markers and RNA was collected in duplicates, except for the E4hi condition which was collected in triplicates. RNAseq was performed for each replicate.biostudies-arrayexpressSample Collection - Mammary glands, uterus and vagina were harvested from ER-rtTA/TetOCre/RosaYFP mice and rinsed in PBS. Uterus and vagina were chopped before being incubated in collagenase from clostridium histolyticum (300U/ml, C0130-5G, Sigma) and hyaluronidase from bovine testes (300 μg/ml, H3884-1G, Sigma) for 1h30 at 37°C on a rocking plate. Collagenase enzyme was blocked by the addition of EDTA (5 mM), and cell suspension was rinsed in HBSS supplemented with 10% FBS before filtration throughout a 70-μm cell strainer (BD Bioscience). Samples were rinsed with PBS supplemented with 2% FBS (FACS buffer) and incubated with coupled antibodies diluted at 1/100 in FACS buffer. Antibodies used for uterus and vagina: LIN antibodies comprised rat PE-conjugated anti-CD31 (clone MEC 13.3, 102508, Biolegend) and rat PE-conjugated anti-CD45 (clone 30-F11, 103106, Biolegend), rat APC-Cy7-conjugated anti-CD90.2 (clone 53-2.1, 561641, BD), rat BV711-conjugated anti-Epcam (clone G8.8, 563134, BD). Mammary glands were chopped before being incubated in collagenase (300 U/mL) and hyaluronidase (300 μg/ml) for 2h30 at 37°C on a rocking plate. EDTA was added for 2 minutes to block collagenase and trypsin was added for 2 minutes before cell suspension wash in DMEM medium supplemented with 10% FBS and filtration through 70-μm cell strainer. Samples were rinsed in FACS buffer before incubation in 250 μl per million cells in primary antibody dilution (1/100) for 30 minutes on ice, with shaking every 10 minutes. Antibodies used for mammary gland staining: LIN antibodies comprised: APC-conjugated rat anti-CD45 (clone 30-F11, 17-0451, ebioscience), APC-conjugated rat anti-CD31 (clone 390, 17-0311, ebioscience) and APC-conjugated rat anti-CD140a (clone APA5, 171401, ebioscience), PECy7-conjugated rat anti-CD24 (clone M1/69, 560536, BD), AF700-conjugated Armenian Hamster anti CD29 (clone HMB1-1, 102218, Biolegend/IMTEC), PE-conjugated rat anti-CD49b (clone DX5, 553858, BD) and PerCpCy5-conjugated rat anti-Sca1 (clone D7, 108124, Biolegend/IMTEC), BV421-conjugated rat anti CD140a (clone APA5, 562774, BD), APCCy7-conjugated rat anti CD45 (clone 30-F11, 103116, Biolegend), APCCy7-conjugated rat anti CD31 (clone 390, 102440, Biolegend). After cell suspension staining, samples were washed with FACS buffer and filtered throughout a 40-μm cell strainer (BD Bioscience) before FACS cell sorting. Cell sorting was performed on a FACS Aria III using the FACS DiVa software (BD Biosciences). For BrdU detection by FACS in the mammary gland, the APC BrdU Flow Kit (BD Pharmingen) was used according to the manufacturer’s recommendations.Sample Treatment - To target YFP expression, 4 week old ER-rtTA/TetOCre/RosaYFP female mice were induced with doxycycline diluted in drinking water (2g/L, AG Scientific) during 4 weeks. ER-rtTA/TetOCre/RosaYFP mice or C57BLACK/6J mice were bilaterally ovariectomized at 10 weeks old. E2 and E4 were diluted in 10% ethanol (VWR chemicals) and propylene glycol (Sigma Aldrich). Estrogenic treatments were continuously administered in mice at 12 weeks old for 14 days, thanks to the subcutaneous insertion of Alzet® osmotic minipumps (#2002, Charles River, France) filled with either E2 (0,08 mg/kg/day, Sigma) or E4 (0,3 mg/kg/day or 3 mg/kg/day, gift from Mithra pharmaceuticals). For the ovariectomized condition, mice were ovariectomized at 10 weeks old and sham surgery was performed at 12 weeks old. For short estrogen exposure experiment, ER-rtTA/TetOCre/RosaYFP female were OVX at 10 weeks old and a single intraperitoneal injection of either E2 (0,08 mg/kg/day), E4 (0,3 mg/kg/day or 3 mg/kg/day) or Propylene glycol mixed with 10% EtOH was administered 24 hours before the end of the experiment.Library Construction - Quality of RNA was evaluated by Bioanalyzer 2100 (Agilent). Indexed cDNA libraries were obtained using the Ovation Solo RNA-seq Systems (NuGen) following manufacturer’s recommendations.Nucleic Acid Extraction - RNA was extracted 10 000 - 200 000 cells from sorted cells using RNeasy micro kit (QIAGEN) according to the manufacturer’s recommendations.Sequencing - The multiplexed libraries (11pM/18pM) were loaded on flow cells and sequences were produced using a HiSeq PE Cluster Kit v4 and TruSeq SBS Kit v3-HS (250 cycles) on a Novaseq 6000 (Illumina).OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesSequence Alignment - Reads were mapped against the mouse reference genome (Grcm38/mm10) using STAR software22 to generate read alignments for each sample. Annotations Grcm38.87 were obtained from ftp.Ensembl.org. After transcripts assembling, gene level counts were obtained using HTseq-count23 using options -f bam -r name -s no --nonunique allData Transformation - Raw read counts from mammary gland were imported into R (v4.3.0) and analyzed using DESeq224(v1.42.0). The input matrix contained gene-level counts for experimental samples, with associated metadata containing sample ID, cell type, treatment, and batch information loaded from a corresponding annotation table. Lowly expressed genes were filtered out, retaining only those with at least ten reads across all samples. Library size normalization was performed using the estimateSizeFactors() function to account for sequencing depth differences between samples. Variance-stabilizing transformation (VST) was then applied to the normalized count matrix by using vst(dds, blind = FALSE) function, providing homoscedastic, approximately normally distributed expression values suitable for downstream visualization and clustering. To correct for technical batch effects, the transformed expression matrix was processed using limma25 (v3.26) removeBatchEffect() function, specifying the batch variable extracted from the sample metadata. The batch-corrected expression matrix was exported as a data table for further analysis.UnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq 6000RNA-seq of coding RNAMus musculusYura SongAlexandra Van KeymeulenfalseRNA-seq of murine mammary epithelial cells from ovariectomised females treated with estradiol (E2) or estetrol (E4)To mimick menopause hormone therapy and compare the effects of estetrol (E4) to the ones of estradiol (E2), ovariectomized mice were treated for 2 weeks with E2, a physiological dose of E4 (E4low) or a supra physiological dose of E4 (E4hi). Non treated ovariectomized mice were used as control. Basal cells, estrogen positive luminal cells and estrogen negative luminal cells from MG were sorted based on surface markers and RNA was collected in duplicates, except for the E4hi condition which was collected in triplicates. RNAseq was performed for each replicate.2026-06-01T00:00:00Z2026-06-01T12:54:56.432Z2025-11-13T22:01:42.619ZE-MTAB-16111ERP184404EFO_0002944EFO_0004170EFO_0004917EFO_0005518EFO_0003816EFO_0003738EFO_0004184EFO_0003969