organism: Homo sapiens
description: Protocols: All frozen primary human breast tissue was obtained from the Breast Cancer Now Tissue bank (REC 15/EE/0192). Prior to freezing, fresh tissues were minced and digested O/N in 1 mg/mL Collagenase 1A and 1 mg/mL Hyaluronidase in an orbital shaker, washed 2-3 times and sedimented to allow for the separation between epithelial-enriched and stromal fractions, which were then separately cryostored. Frozen vials of epithelial-enriched or stromal-enriched fractions were defrosted, washed in PBS and centrifuged at 400 g for 5 minutes and resuspended in 2 mL of freshly prepared PBS + 0.025% Trypsin and 0.4 mg/mL Deoxyribonuclease 1 (DNase). Samples were then incubated at 37°C with pipetting up and down for 2-3 minutes until smoothly digested or up to a maximum of 10 minutes. Next, samples were washed and centrifuged for 20 minutes at 400 g with slow break. The pellet was resuspended in media + DNase until homogeneous, then diluted and filtered through a 40 μm cell strainer. After centrifugation for 5 minutes at 400 g, the pellet was resuspended by pipetting up and down in 200 μL of Cell Preparation Medium + 10 μL of 10 mg/mL DNase until homogeneous, then washed in 3-6 mL of media. 30,000 cells were resuspended into 48 μL of media and 400 Human mammary epithelial cells (HuMECs, Thermo Fisher Scientific A10565) were added as spike-in, and samples were submitted for scRNA-seq (either stromal-enriched or epithelial-enriched samples). For the epithelial-enriched fraction only, the rest of the processed sample was stained with primary antibodies: CD45-APC, CD31-APC, EPCAM-AF488, CD49f-PE/Cy7. DAPI was used to detect dead cells. Cells were filtered through a cell strainer before sorting. Sorting of cells was done using a FACS Aria Fusion sorter. Single-stained control cells were used to perform compensation manually and unstained cells were used to set gates. After doublets, dead cells and contaminating haematopoietic and endothelial cells (referred to as lineage) were gated, up to 30,000 LASPs were sorted for scRNA-seq (with the addition of 400 HuMECs as spike-in). Approximately 30,000 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).
attributes.tag: ENA first public
attributes.value: 2024-04-09
attributes.tag: External Id
attributes.value: SAMEA115129848
attributes.tag: INSDC center name
attributes.value: Cambridge Stem Cell Institute Department of Pharmacology
attributes.tag: INSDC last update
attributes.value: 2024-01-23T22:17:35Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-13664_4:D4_Epi_all_SLX-19864_SIGAD7
attributes.tag: age
attributes.value: 25
attributes.tag: batch
attributes.value: 15/10/2020
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: collection date
attributes.value: not collected
attributes.tag: common name
attributes.value: human
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: disease
attributes.value: normal
attributes.tag: fraction
attributes.value: Epithelial enriched - unsorted
attributes.tag: genotype
attributes.value: assume_WT
attributes.tag: geographic location (country and/or sea)
attributes.value: not collected
attributes.tag: individual
attributes.value: Donor_4
attributes.tag: isolate
attributes.value: not applicable
attributes.tag: organism part
attributes.value: breast
attributes.tag: parity
attributes.value: 0
attributes.tag: protocol
attributes.value: 10x 3' v3
attributes.tag: sample id
attributes.value: SLX-19864_SIGAD7
attributes.tag: sample name
attributes.value: E-MTAB-13664_4:D4_Epi_all_SLX-19864_SIGAD7
attributes.tag: scientific_name
attributes.value: Homo sapiens
attributes.tag: sex
attributes.value: female
attributes.tag: surgery type
attributes.value: reduction mammoplasty