characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2020-05-23T04:03:34Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2019-10-09T16:41:40Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA6061941
characteristics.characteristic: External Id
characteristics.text: Program in Genetics and Genome Biology, SickKids Research Institute Department of Molecular Genetics, University of Toronto
characteristics.characteristic: INSDC center name
characteristics.text: 2020-05-23T04:03:34Z
characteristics.characteristic: INSDC first public
characteristics.text: 2019-10-09T16:41:40Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS3863066
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-8394:LuVEC-iPL lung scaffold biorep 3
characteristics.characteristic: Submitter Id
characteristics.text: 12
characteristics.unit: week
characteristics.characteristic: age
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: induced into progenitor-like cell - LuVEC-iPL
characteristics.characteristic: cell type
characteristics.text: house mouse
characteristics.characteristic: common name
characteristics.text: Protocols: F1 littermates of C56B/6 mouse crossed with R26 rtTA :Col1a1 4F2A double transgenic mouse were used in this experiment. To isolate lung vascular endothelial cells, mice lungs were perfused with 5mL of ice cold PBS via the right ventricle with the use of a 27G needle. The trachea was isolated and 2 mL of Dispase II solution (2.0 U/mL; Roche Applied Science, Germany) was instilled through the tracheal cannula. After the ligation of the trachea, the lung was incubated in a 50 mL conical tube containing 8 mL of Dispase II solution (2.0 U/mL) at 37ºC for 30 minutes using a shaking incubator. Then lungs were isolated from the heart, thoroughly minced and incubated further for 15 minutes in a enzyme mix containing 8 mL of Dispase II, 1 mL of Collagenase/Dispase (final concentration 1 mg/mL; Roche Applied Science, Germany) and 1 mL of DNase I (final concentration 0.1 mg/mL; Sigma). After removing red blood cells by RBC lysis buffer (Sigma), cells were resuspend in PBS containing MACS Mouse CD45 MicroBeads (Miltenyi Biotec, Germany) for 15 minutes at 4ºC. The cells were then applied to the AutoMACS cell separator system (Miltenyi Biotec) to deplete CD45+ hematopoietic cells. The recovered CD45- cells were treated with MACS Mouse CD31 MicroBeads (Miltenyi) and subsequently subjected to the AutoMACS cell separator system to isolated CD31+ cells.To isolate liver sinusoidal endothelial cells, mice livers were excised and cut into small pieces and incubated with 0.1% collagenase A in DMEM for 1.5 hours in a shaker incubator at 37ºC. The cells were pelleted and resuspend in RBC lysis buffer (Sigma) followed by incubation with mouse CD146 (LSEC) microbeads (Miltenyi). LSEC separation was performed using AutoMACS system (Miltenyi) according to the manufacturer’s instruction. Isolated cells were cultured on 2% gelatin (Sigma-Aldrich, Canada) coated culture plates at a cellular density of 1500 cell/mm2 with Embryonic Stem Cell media (ES media) composed of knockout DMEM (Thermo Fisher Scientific), 15% of ES qualified FBS, 1000UI of mouse recombinant Leukemia Inhibitory Factor. Mouse-scale perfusion-based bioreactor was assembled for gravity-driven cell injection. A cell reservoir was placed 30 cm above the organ chamber in which a decellularized lung was connected through the PA cannula. A pinch valve (Takasago Fluidics, Japan) was controlled to open for 0.5-second every 0.5-second. Twenty millions of cells were resuspended in 40 mL of EC media in a 100 mL glass bottle and the typical injection rate was 1 mL/min. After cell injection, the media circuit was opened. Media flow was generated by a Masterlfex L/S Precision Modular Drivers with Bench Top Controller (Cole-Parmer, Canada) at a rate of 0.3 ml/min for 3 days. The media was replaced every other day. Cells were incubated in ES media plus doxycycline (1.5 µg/mL) for 21 days. RNA was purified from LuVECs and LSECs (n=3 biological replicates per condition: primary, induced, and scaffold-injected) using RNeasy Mini Kit (Qiagen). RNA quality was assessed with Agilent 2100 Bioanalyzer. mRNA-seq libraries were prepared using TruSeq Stranded mRNA Library Prep kit (Illumina)
characteristics.characteristic: description
characteristics.text: R26 rtTA :Col1a1 4F2A heterozygote
characteristics.characteristic: genotype
characteristics.text: recellularized on lung scaffolds in EC media
characteristics.characteristic: growth condition
characteristics.text: 3
characteristics.characteristic: individual
characteristics.text: Mus musculus
characteristics.characteristic: organism
characteristics.text: Mus musculus
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_10090
characteristics.characteristic: organism
characteristics.text: lung blood vessel
characteristics.characteristic: organism part
characteristics.text: doxycycline induced 4F2A transgene expression
characteristics.characteristic: phenotype
characteristics.text: lung vascular endothelial cell (LuVEC)
characteristics.characteristic: progenitor cell type
characteristics.text: Mus musculus
characteristics.characteristic: scientific_name
characteristics.text: male
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/PATO_0000384
characteristics.characteristic: sex
characteristics.text: C56B/6
characteristics.characteristic: strain
characteristics.text: LuVEC-iPL lung scaffold biorep 3
characteristics.characteristic: title