characteristics: ERC000011
characteristics: ENA-CHECKLIST
characteristics: 2022-10-20T00:17:58Z
characteristics: ENA-FIRST-PUBLIC
characteristics: 2022-10-20T00:17:58Z
characteristics: ENA-LAST-UPDATE
characteristics: SAMEA111452527
characteristics: External Id
characteristics: Department of Cell and Developmental Biology, Department of Internal Medicine, Department of Biomedical Engineering Medicine, University of Michigan Medical School, Ann Arbor, MI
characteristics: INSDC center name
characteristics: 2022-10-20T00:17:58Z
characteristics: INSDC first public
characteristics: 2022-10-20T00:17:58Z
characteristics: INSDC last update
characteristics: public
characteristics: INSDC status
characteristics: ERS13546786
characteristics: SRA accession
characteristics: E-MTAB-12266:HT328-ESO_HYENAC
characteristics: Submitter Id
characteristics: 63
characteristics: year
characteristics: age
characteristics: ArrayExpress
characteristics: broker name
characteristics: human
characteristics: common name
characteristics: Protocols: Biopsies are collected in complete HYENAC cold medium at the University Hospital. Samples are process for single-cell dissociation, tissue fixation and in vitro culture within a 24hr timepoint of sample collection. To dissociate patient biopsies for scRNA-seq, tissue was placed in a Petri dish with ice-cold 1× HBSS (with Mg2+, Ca2+). To prevent adhesion of cells, all tubes and pipette tips were pre-washed with 1% bovine serum albumin (BSA) in 1× HBSS. The tissue was minced manually using springsqueeze scissors before being transferred to a 15 ml conical flask containing 1% BSA in HBSS. Tubes were spun down at 500 g for 5 min at 10°C, after which excess HBSS was aspirated. Mix 1 from the Neural Tissue Dissociation Kit (Miltenyi, 130-092-628) containing dissociation enzymes and reagents was added and incubated at 10°C for 15 min. Mix 2 from the Neural Tissue Dissociation Kit was added, and the suspension was fluxed through P1000 pipette tips, interspersed with 10 min incubations at 10°C. Flux steps were repeated as needed until cell clumps were no longer visible under a stereomicroscope. Cells were filtered through a 1% BSAcoated 70 μm filter using 1× HBSS, spun down at 500 g for 5 min at 10°C, and resuspended in 500 μl 1× HBSS (with Mg2+, Ca2+). Then 1 ml of RBC Lysis Buffer (Roche, 11814389001) was added, and tubes were incubated on a rocker at 4°C for 15 min. Cells were spun down at 500 g for 5 min at 10° C, then washed twice in 2 ml 1% BSA, being spun down at 500 g for 5 min at 10°C each time. A hemocytometer was used to count cells, and samples were then spun down and resuspended to reach a concentration of 1000 cells/μl and kept on ice. Tissue samples are minced and/or dissociated into single-cells. They are co-culture with sublethally irradiated fibroblast in complete HYENAC medium. Upon reaching 60-80% confluency, cultures are typsinized and passage. Immediately after tissue dissociation, single cell libraries were prepared on the 10x Chromium with a target of 5,000 cells. Chromium Single Cell 3’ Library & Gel Bead Kit v2, 16 rxns PN-120237 was used. Single-cell libraries were prepared using the Chromium Next GEM Single Cell 3′ Library Construction Kit v3.1 according to the manufacturer’s instructions.
characteristics: description
characteristics: adult
characteristics: developmental stage
characteristics: Barrett's esophagus
characteristics: disease
characteristics: 2D esophagus cells expanded in 3T3-J2i feeder layer; complete HYENAC+CBS
characteristics: growth condition
characteristics: HT328
characteristics: individual
characteristics: Homo sapiens
characteristics: organism
characteristics: Homo sapiens
characteristics: organism
characteristics: esophagus
characteristics: organism part
characteristics: normal tissue adjacent to lesion
characteristics: sampling site
characteristics: Homo sapiens
characteristics: scientific_name
characteristics: female
characteristics: sex
characteristics: HT328-ESO_HYENAC
characteristics: title