Protocols: All tubes and pipette tips were pre-washed with 1% BSA in 1X HBSS (all HBSS in protocol is with Mg2+ and Ca2+) to prevent cell adhesion to the plastic. The tissue was placed in a petri dish in ice-cold 1X HBSS, and the tissue was minced under a stereomicroscope using scissors. For uncultured lung tissue, roughly 1cm2 of the most distal portion of the lung was isolated, and for lung explants, 4 explants were collected per condition. The whole explants were used for sequencing. The minced tissue was transferred to a 15mL conical tube with the HBSS, centrifuged at 500g for 5 minutes at 10ºC, and the HBSS was removed. Mix 1 from the Neural Tissue Dissociation Kit (Miltenyi, Cat#130-092-628) was added to each tube, and the tube was placed at 37ºC for 15 minutes, then Mix 2 was added and the cells were incubated for 10 minutes at 37ºC. The cells were agitated by harshly pipetting with a P1000. The incubation/agitation step was repeated every 10 minutes until the cells looked to be a single cell suspension, approximately 30 minutes. The cells were then filtered through a 70µm filter, pre-coated with 1% BSA in 1X HBSS, into a 15mL conical tube. The filter was rinsed 3x with 1mL 1% BSA in 1X HBSS. The cells were centrifuged at 500g for 5 minutes at 10ºC, and the supernatant was removed. 1mL Red Blood Cell Lysis Buffer (Sigma, Cat#11814389001) and 0.5mL 1% BSA in 1X HBSS was added to the tube, and the tube was rocked for 15 minutes at 4ºC. The cells were centrifuged at 500g for 5 minutes at 10ºC, washed twice in 2mL 1% BSA in 1X HBSS and centrifuged again. The cells were resuspended in 200µL 1% BSA in 1X HBSS, counted using a hemocytometer, centrifuged at 500g for 5 minutes at 10ºC, and resuspended to reach a concentration of 1,000 cells/µL. For air-liquid-interface culture, Nucleopore Track-Etched Membranes (13mm, 8µm pore, polycarbonate) (Sigma, Cat#WHA110414) placed in 24-well tissue culture plates (Thermo Fisher, Cat#12-565-163) were pre-coated with 20µg/cm2 Collagen Type I (Sigma, Cat#C5533) in 0.01N ice-cold acetic acid for 30 minutes on ice followed by 2 hours at 37ºC. The membranes were then washed with 1X PBS directly before use. To prepare the explants, the lung was placed in a petri dish in ice-cold 1X PBS and approximately 1mm2 pieces of tissue were cut from the most distal edge of the lung under a stereomicroscope using forceps and a scalpel. 500µL culture media containing Advanced DMEM/F-12 (Thermo Fisher, Cat#12634010), 100µg/mL penicillin-streptomycin (Thermo Fisher, Cat#15140122), 2mM L-Glutamine (Thermo Fisher, Cat#25030081), 10mM HEPES (Corning, Cat#25060CI), 1 bottle B-27 Supplement (Thermo Fisher, Cat#17504044), 1 bottle N-2 Supplement (Thermo Fisher, Cat#17502048), and 0.4µM 1-Thioglycerol (Sigma, Cat#M1753) was added to each well in the plate underneath the membrane. One explant per membrane was placed directly on the membrane in the center. Media was changed every 2 days. Immediately following placement of the explants on the Nucleopore Track-Etched Membranes, 10^10 pfu of control or LGR5 ECD adenovirus previously described in Yan et al. (Yan et al., Nature, 2017) was pipetted directly on top of each explant under a stereo microscope using a p10 pipette. A maximum of 2µL adenovirus was added to each explant at a time to prevent the adenovirus from running off the explant. The explants were re-infected every 2 days. Infection of the tissue was confirmed by immunofluorescence for FLAG and the murine IgG2a Fc fragment for the LGR5 ECD adenovirus and control adenovirus respectively. Immediately after tissue dissociation, single cell libraries were prepared on the 10x Chromium with a target of 5000 cells. Chromium Single Cell 3' Library & Gel Bead Kit v2, 16 rxns PN-120237 was used Immediately after tissue dissociation, single cell libraries were prepared on the 10x Chromium with a target of 5000 cells. Chromium Single Cell 3' Library & Gel Bead Kit v2, 16 rxns PN-120237 was used
ENA-FIRST-PUBLIC
2022-01-01T00:12:56Z
ENA-LAST-UPDATE
2022-01-01T00:12:56Z
External Id
SAMEA9312521
INSDC center name
University of Michigan Health System - Internal Medicine - Gastroenterology
INSDC first public
2022-01-01T00:12:56Z
INSDC last update
2022-01-01T00:12:56Z
INSDC status
public
Submitter Id
E-MTAB-10662:HFL-explant_control-virus
age
13
broker name
ArrayExpress
cell type
various
common name
human
developmental stage
late embryonic stage
disease
normal
growth condition
in vitro explant
individual
1
infect
control adenovirus
organism part
lung
sample name
E-MTAB-10662:HFL-explant_control-virus
sampling site
distal lung
scientific_name
Homo sapiens
sex
female