characteristics.text: 2024-01-01
characteristics.characteristic: ENA first public
characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: SAMEA115057726
characteristics.characteristic: External Id
characteristics.text: Department of Internal Medicine (Nephrology) & Einthoven Laboratory of Vascular and Regenerative Medicine, Leiden University Medical Center, Leiden, The Netherlands
characteristics.characteristic: INSDC center name
characteristics.text: 2023-12-22T13:48:00Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS17598222
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-13671:PECAR1044_L291
characteristics.characteristic: Submitter Id
characteristics.text: 3
characteristics.unit: month
characteristics.characteristic: age
characteristics.text: 1
characteristics.characteristic: batch
characteristics.text: ArrayExpress
characteristics.characteristic: broker name
characteristics.text: endothelial cell
characteristics.characteristic: cell_type
characteristics.text: not collected
characteristics.characteristic: collection date
characteristics.text: house mouse
characteristics.characteristic: common name
characteristics.text: Protocols: Twenty-four hours after renal ischemia-reperfusion injury, sham and injured wild-type and Oxct1 knock-out mice receiving control or ketogenic diets were anesthetized with an intraperitoneal injection of ketamine/xylazine (Dechra, Innovet) and blood was collected from the right ventricle. Mice were then submitted to left heart perfusion with 3ml PBS (1ml/min) and perfused kidneys were collected in ice-cold HBSS. Kidney capsules were removed, and kidneys were cut into small pieces in a collagenase digestion buffer (2 mg/ml collagenase I [C9891; Sigma-Aldrich], 27 IU/ml DNAse I [D4527; Sigma-Aldrich], and HBSS [14025100; Thermo Fisher Scientific]). The samples were then incubated for 40 minutes at 37°C, shaking every 10 minutes, and digestion was stopped by adding ice-cold HBSS. Digested samples were filtered on a 40 mm cell strainer, immune cells were separated using CD45 magnetic microbeads and QuadroMACS separators with MS columns (Miltenyi Biotec). Endothelial cells were then enriched from the CD45- population using CD31 magnetic microbeads and QuadroMACS separators with MS columns (Miltenyi Biotec). Cell populations were resuspended in FACS buffer (0.1% BSA, 2 mM EDTA in PBS) and stained for flow cytometry analysis and sorting. Upon magnetic sorting, CD31+ fraction was incubated for 30min at 4°C with: CD45 (1/100, 561047; BD Pharmingen), CD31 (1:50, 130-111-355; Miltenyi Biotec) and viability dye (1/1000, 65-0865-14; Invitrogen). OneComp ebeads (01–1111–42; Thermo Fisher Scientific) and cooked cells (20 minutes at 60°C) were used for compensation. Viable endothelial cells (live CD45-CD31+ ECs) were FACS-sorted using a FACS Aria III (BD Biosciences), and collected in wash buffer (0.1% BSA in PBS). Cells were centrifuged for 6 minutes at 400xg, and resuspended in 350 ul of RLT buffer supplemented with DTT (20 ul of 2 M DTT in 1 ml RLT buffer). Samples were stored at -80°C until RNA extraction. To obtain a tamoxifen-inducible, EC-specific Oxct1 knock-out mouse line, Oxct1lox/lox mice (provided by P. Crawford) were intercrossed with VECadh-CreERT2 mice (provided by T. Mäkinen) generating VECadh-CreERT2;Oxct1lox/lox C57BL/6N mice. On subsequent intercrossing with double fluorescent mT/mG Cre reporter mice C57BL/6J, the resulting VECadhΔOXCT1:mTmG mice allowed determination of recombination efficiency after tamoxifen treatment. Tamoxifen (75 mg/kg) was administered for 5 consecutive days to 7- to 8-week-old male mice by intraperitoneal injection and experiments were started 1 week later. Before IRI or sham procedure, mice were then exposed to 3-weeks diet pre-treatment with control diet (V1535-000; Ssniff) or ketogenic diet (F3666; Plexx) with ad libitum access to food and water. For the renal ischemia/reperfusion injury experiment, Mice were anesthetized with isofluorane and placed on a heating pad (37°C) to maintain constant body temperature. An incision was performed on the back to expose the renal pedicles. A bilateral clamping was performed for 30min, then the clamping was released, and reperfusion was allowed. After suture of the wound, mice were allowed to recover, and Buprenorphine was administered (1mg/kg). Total RNA was extracted from freshly isolated murine RECs using RNeasy® Micro Kit (Qiagen) according to the manufacturer’s instructions. RNA quality and quantity were measured with NanoDrop (Thermo Fisher Scientific). Libraries were prepared using the QuantSeq 3' mRNA-seq Library Prep Kit FWD for Illumina (Lexogen) adjusting the number of cycles for amplification to the RNA input quantities according to manufacturer’s instruction. Optimal cycle number was determined by using PCR Add-on kit for Illumina (Lexogen).
characteristics.characteristic: description
characteristics.text: adult
characteristics.characteristic: developmental stage
characteristics.text: chow diet
characteristics.characteristic: diet
characteristics.text: wild type genotype
characteristics.characteristic: genotype
characteristics.text: not collected
characteristics.characteristic: geographic location (country and/or sea)
characteristics.text: L291
characteristics.characteristic: individual
characteristics.text: ischemia reperfusion injury
characteristics.characteristic: injury
characteristics.text: not applicable
characteristics.characteristic: isolate
characteristics.text: Mus musculus
characteristics.characteristic: organism
characteristics.text: Mus musculus
characteristics.characteristic: organism
characteristics.text: kidney
characteristics.characteristic: organism part
characteristics.text: Mus musculus
characteristics.characteristic: scientific_name
characteristics.text: male
characteristics.characteristic: sex
characteristics.text: C57Bl6/N
characteristics.characteristic: strain
characteristics.text: PECAR1044_L291
characteristics.characteristic: title