2024-04-09
ENA first public
ERC000011
ENA-CHECKLIST
SAMEA115129810
External Id
Cambridge Stem Cell Institute Department of Pharmacology
INSDC center name
2024-01-23T22:17:35Z
INSDC last update
public
INSDC status
ERS17832493
SRA accession
E-MTAB-13664_4:D38_Epi_all_SLX-20261_SIGAA3
Submitter Id
39
year
age
26/01/2021
batch
ArrayExpress
broker name
not collected
collection date
human
common name
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).
description
adult
developmental stage
normal
disease
Epithelial enriched - unsorted
fraction
BRCA2
genotype
not collected
geographic location (country and/or sea)
Donor_38
individual
not applicable
isolate
Homo sapiens
organism
Homo sapiens
organism
breast
organism part
0
parity
10x 3' v3
protocol
SLX-20261_SIGAA3
sample id
Homo sapiens
scientific_name
female
sex
prophylactic mastectomy
surgery type
D38_Epi_all_SLX-20261_SIGAA3
title