GSM9081252: Sample 5 - Endometrial Cells; Homo sapiens; RNA-Seq
Endometrial samples were either fixed in 4% PFA overnight at 4C (n=3) or placed into cold DMEM:F12 collection medium supplemented with 5% newborn calf serum and antibiotics/antimycotic and immediately transported at 4C to the laboratory for processing (n=5). Endometrial tissue was digested to single cells within 18 hours. The functionalis was removed by scraping the tissue off the surface of the myometrium and finely minced, then the full basalis was recovered by dissecting 1-2 mm myometrium with attached endometrium (visible microscopically) and finely dicing the tissue.The minced tissue underwent a 2-step enzymatic/mechanical dissociation with vigorous trituration every 5 min at 37C in PBS (Gibco) containing collagenase type I (5 mg/mL; Worthington), DNAse I (0.08 mg/mL; Worthington) and glucose (5mM; Merck), then strained (40 µM sieve, Falcon) to separate  stromal cells from epithelial gland fragments. Red blood cells in the stromal fraction were removed by Ficoll-paque (Sigma-Aldrich) density gradient separation. Epithelial cells underwent a second digestion with a similar cocktail using collagenase type II (0.8 mg/ml, Worthington) and DNAse I 0.16 mg/mL, then epithelial cells were strained using a 40 µM sieve. The single cell fractions were counted and combined for FACS staining. Single cell suspensions were blocked with Fc Receptor blocker (FcR; 2 µL/106 cells, Miltenyi Biotec) and Rat IgG (4.4 µg/106 cells; Jackson ImmunoResearch) as described (Wyatt et al., 2021). Cells were then distributed in tubes as unstained, viability and Fluorescence minus 1 (FMO) controls (up to 5X105 cells in 100 µL) and the full antibody panel (remaining cells at 5X105/100µL at the same final antibody concentration) and incubated with conjugated antibodies, PE-CD325 (N-cadherin) (10 µg/mL), BV421-CD15 (SSEA1) (1.25 µg/mL; BioLegend), APC-SUSD2 (1.25 µg/mL; BioLegend,), BB515-CD326 (EpCAM) (0.25 µL/5X105cells, BD Biosciences) and CD45 (pan leukocyte exclusion marker) PECy7-CD45 (0.25 µL/5X105cells, BD Biosciences), for 25 min at 4ºC. Propidium iodide (PI; 62.5 ng/mL; BD Biosciences) was added 10 min before sorting (Wyatt et al., 2021). Compensation Plus Particle Set beads (60 µL/test; BD Biosciences) were stained as single staining protocols with the above antibodies following manufacturer's instructions. Labelled endometrial cells underwent FACSorting of endometrial stem/progenitor cell subpopulations and their differentiated progeny (epithelial (EpCAM+) progenitors N-cadherin+/SSEA1-; N-cadherin+/SSEA1+; N-cadherin-/SSEA1+; mature epithelial cells, EpCAM+/N-cadherin-/SSEA1-) (Nguyen et al., 2017) and eMSC (SUSD2+; SUSD2- fibroblasts) (Masuda et al., 2012) using an Influx Cytopeia-BD 5 laser sorter and BD FACS™ Sortware software (Monash University FlowCore). The 6 cell fractions were tagged using TotalSeq A anti-human hashtags. The stem/progenitor cell populations were pooled and mature epithelial and stromal cells added for scRNAseq for each of the 5 biological samples to generate barcoded single-cell 3′ gene expression libraries with the Chromium Single-cell 3′ GEM, Library & Gel Bead Kit v3 (10x Genomics) and their matched HTO library to resolve the hashtags (BioLegend). The quality of the libraries was assessed by Agilent Bioanalyzer High Sensitivity DNA chip. Libraries were sequenced to an average depth of 384,676 reads/cell on the Illumina NovaSeq6000 at UQ. Sequence quality checks revealed that HTO library construction had failed; and were excluded from subsequent analyses.
GEO
GSM9081252_r1