biostudies-arrayexpressBruno SimoesHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-13819Vacuum-assisted biopsy (VAB) from normal breast tissue were obtained from women participating in the Breast Cancer - Anti-Progestin Prevention Study 1 (BC-APPS1). A baseline VAB was performed in the luteal phase of the menstrual cycle and a second VAB was performed after a 12-week anti-progestin (ulipristal acetate, daily 5mg oral tablet) treatment. Breast tissue minced into small fragments was digested overnight with collagenase/hyaluronidase. Following further digestion with trypsin and dispase, a dissociated breast single cell suspension was obtained and single-cell RNA-sequencing was performed. Standard workflow for the 10X Genomics Single Cell 3’ RNA Kit was used in two batches of 6 samples (3 participants per run with pre and post-ulipristal acetate treatment).biostudies-arrayexpressNucleic Acid Extraction - Approximately 10,000 mammary cells per sample were loaded into separate channels of a single Chromium controller (10x Genomics) chip. Consequently, gel bead-in-emulsions (GEMS) were generated using 10X Genomics Single Cell 3’ Reagent Kit v3.Sample Collection - Normal breast tissue obtained via Vacuum-Assisted Biopsy (VAB) was manually minced with a scalpel into small fragments (approximately 2mm pieces) and incubated in dissociation medium: phenol red free DMEM/F12 with HEPES (Gibco) supplemented with 25% BSA Fraction V solution (Gibco), 1mg/mL collagenase/hyaluronidase (Stem Cell Technologies) and 5μg/mL insulin (Sigma). After overnight digestion at 37°C with shaking at 100 rpm, the dissociated breast cell suspension was centrifuged at 450 x g for 5 minutes at 4°C. The fat layer was discarded and the epithelial pellet was resuspended in DMEM/F12 medium and centrifuged again. This wash step was repeated until the supernatant became clear. Then, 1 mL of pre-warmed 0.05% Trypsin-EDTA was added to the enriched epithelial pellet pipetting it up and down gently with a P1000 pipette for 2 - 3 minutes. Next, 10mL of cold Hank’s Balanced salt solution (HBSS, Gibco) supplemented with 10mM Hepes (Gibco) and 2% FBS (Gibco) was added and the cells were centrifuged at 450 x g for 5 minutes at 4°C. After removing the supernatant, 1ml of pre-warmed 5 mg/ml dispase (StemCell Technologies) was added to the sample and pipetted for 1 minute to further dissociate cell clumps. Cells were resuspended in HBSS/Hepes/FBS solution, centrifuged and supernatant was discarded. HBSS/Hepes/FBS solution was then added and cells were sieved using 100μm and 40μm filters to yield a single cell suspension. Cells were counted using a Fuchs Rosenthal haemocytometer and frozen using Bambanker freezing media (Lymphotec Inc.) until single cell RNA-sequencing.Library Construction - Sequencing libraries were prepared using Single Cell 3’ Reagent Kits v3 (10X Genomics) and then converted using the NovaSeq 6000 S2 Reagent Kit (Illumina).Sequencing - Sequencing of single cell libraries was performed using a NovaSeq 6000 instrument using a S2 flow cell.Sample Treatment - A baseline VAB was performed in the luteal phase of the menstrual cycle and a second VAB was performed after a 12-week anti-progestin (ulipristal acetate, daily 5mg oral tablet) treatment.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesSequence Alignment - Data was processed using Cell Ranger version 3.0.2 (10x Genomics) using the human genome GRCh38 as a reference.Data Transformation - Data from cellranger was imported and clean count expression matrices were generated using the “DropletUtils” R package. A single cell object (sce.rds) was generated, where subsequent filtering and normalization was performed using the “scater” and “scran” R packages. These packages were also used to perform downstream analysis to assign clusters and compute differential gene expression analysis between assigned groups.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq 6000RNA-seq of coding RNA from single cellsHomo sapiensBruno SimoesAlecia-Jane TwiggerfalseSingle cell RNA-sequencing of normal breast tissue samples before and after 12-week anti-progestin (ulipristal acetate) treatmentVacuum-assisted biopsy (VAB) from normal breast tissue were obtained from women participating in the Breast Cancer - Anti-Progestin Prevention Study 1 (BC-APPS1). A baseline VAB was performed in the luteal phase of the menstrual cycle and a second VAB was performed after a 12-week anti-progestin (ulipristal acetate, daily 5mg oral tablet) treatment. Breast tissue minced into small fragments was digested overnight with collagenase/hyaluronidase. Following further digestion with trypsin and dispase, a dissociated breast single cell suspension was obtained and single-cell RNA-sequencing was performed. Standard workflow for the 10X Genomics Single Cell 3’ RNA Kit was used in two batches of 6 samples (3 participants per run with pre and post-ulipristal acetate treatment).2025-09-08T00:00:00Z2025-09-08T01:02:44.503Z2024-02-14T23:55:33.771ZE-MTAB-13819ERP157457EFO_0002944EFO_0004170EFO_0005684EFO_0004917EFO_0005518EFO_0003816EFO_0004184EFO_0003969