title: Single cell RNA-sequencing of normal breast tissue samples before and after 12-week anti-progestin (ulipristal acetate) treatment
design: Single cell RNA-sequencing of normal breast tissue samples before and after 12-week anti-progestin (ulipristal acetate) treatment
library_construction_protocol: 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. 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.  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. Sequencing libraries were prepared using Single Cell 3' Reagent Kits v3 (10X Genomics) and then converted using the NovaSeq 6000 S2 Reagent Kit (Illumina).
identifiers.namespace: European Bioinformatics Institute
identifiers.id: E-MTAB-13819:18T2_p