title: Sample 11
organism: Mus musculus
description: Protocols: The care and use of all mice in this study was carried out in accordance with the UK Home Office regulations, UK Animals (Scientific Procedures) Act of 1986 under the Home Office Project Licences: P1AE9A736, PP4262564, PP708858and LO45CA465. Mice were maintained in 12-h light/12-h dark cycleat 20°C to 22°C, humidity of 40-50%, with ad libitumaccess to water. Mice were fed “light diet” (standard rodent chow) or “heavy diet”; Mouse Express L-Lysine (13C6, 99%; MLK-LYS-C) purchased from CK Isotopes Ltd (UK), as per the labelling experimental design. For the pulse-time experiments, wildtype C57BL/6J mice (Charles River, UK) were employed. For the RNA sequencing experiment and Alport proteomics study, Col4a5−/mice were employed. These mice were obtained from the International Mouse Phenotyping Consortium [doi: 10.1038/nature19356]and the line was generated by deletion of the critical exon 36, resulting in the absence of the collagen-α5(IV) protein, using the published allele map [DOI: 10.1038/ng.3901]. Col4a5-/mice were on a C57BL/6J background. Heterozygous females were crossed with wildtype males and hemizygous male offspring were used to study Alport syndrome and wildtype male littermates were used as a control. All mice gained weight in accordance with reference laboratory data for C57BL/6J mice.  Mice were sacrificed by cervical neck dislocation and kidneys were immediately dissected. The surrounding fat, adrenal glands and renal capsule were removed, and kidneys used for RNA sequencing and proteomic experiments were snap frozen and stored at -80oC. Kidneys used for immunofluorescence imaging were embedded in OCT compound (PFM medical UK Ltd, cat ref #PRC/OCT) and stored at -80oC until cryosectioning. Total RNA was extracted from 30 mg of kidney tissue using TRIzol reagent (Invitrogen). In brief, 1 ml of TRIzol was added to minced kidney tissue and homogenised with a micro pestle. Following phase separation with 0.2 ml of Chloroform, the aqueous phase was mixed with 0.5 ml of isopropanolol and centrifuged for 10 minutes at 12,000 x g at 4 ºC. RNA precipitate was washed with 75% ethanol. RNA in 100 L of RNase-free H2O was cleaned up using RNeasy mini column (QIAGEN) as per manufacturers protocol. RNA was eluted in nuclease-free water and concentration measured using a NanoDrop 2000 (Thermo Fisher Scientific). The quality of all resuspended mRNA was measured using a QubitTM Fluorometer (Thermo Fisher Scientific) and RNA integrity measured using a 2200 TapeStation (Agilent Technologies) to ensure good quality for downstream analysis.  Illumina stranded mRNA library
attributes.tag: ENA first public
attributes.value: 2026-01-22
attributes.tag: INSDC center name
attributes.value: UOM-BCF
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-15467:Sample 11
attributes.tag: age
attributes.value: 16
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: collection date
attributes.value: not collected
attributes.tag: common name
attributes.value: house mouse
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: genotype
attributes.value: col4a5-
attributes.tag: geographic location (country and/or sea)
attributes.value: not collected
attributes.tag: individual
attributes.value: 5
attributes.tag: isolate
attributes.value: not applicable
attributes.tag: organism part
attributes.value: kidney
attributes.tag: sample name
attributes.value: E-MTAB-15467:Sample 11
attributes.tag: scientific_name
attributes.value: Mus musculus
attributes.tag: sex
attributes.value: male
attributes.tag: strain
attributes.value: C57BL/6J