characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2023-06-21T00:18:24Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2023-06-21T00:18:24Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA112890775
characteristics.characteristic: External Id
characteristics.text: CARDIFF UNIVERSITY
characteristics.characteristic: INSDC center name
characteristics.text: 2023-06-21T00:18:24Z
characteristics.characteristic: INSDC first public
characteristics.text: 2023-06-21T00:18:24Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS14896915
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-12845:R087-H-020
characteristics.characteristic: Submitter Id
characteristics.text: 8 to 12
characteristics.unit: week
characteristics.characteristic: age
characteristics.text: ArrayExpress
characteristics.characteristic: broker name
characteristics.text: house mouse
characteristics.characteristic: common name
characteristics.text: Protocols: Wound tissue dissected from 2 mice (8 wounds in total, 4 wounds per mouse) to generate one sample were snap frozen in liquid N2 before being stored at -80 0C. For WT and Alox15-/- a total of four samples were generated at each timepoint Wounds were placed frozen into a pre-cooled pestle and mortar, then ground into a fine powder with liquid N2 added. Tri reagent (1 ml, Sigma-Aldrich, UK) was added. The sample was then transferred to RNAse-free tubes and bromochloropropane (200 µl, Sigma-Aldrich, UK) added before vortexing. Samples were placed on ice for 5 min, before being centrifuged at 15,000 g for 15 min at 4 oC. The upper aqueous phase was transferred to a new tube and 250 µl of 3 M sodium acetate (pH 5.5), 700 µl (100 %) propanol and 10 µl glycogen added, before incubation at -80 0C overnight. Samples were centrifuged at 15,000 g for 15 min at 40 C to pellet the RNA. The pellet was washed 3 times using 70 % ethanol, then allowed to air dry for 5 min. RNAse free water (50 µl) was added. Samples were cleaned using an RNeasy MinElute Cleanup Kit (Catalogue number 74204 Qiagen, MD, USA), using a column based clean up step. Sample (1 µl) was analysed using a NanoDrop™ 2000/2000c Spectrophotometer (ThermoFisher Scientific, Newport UK), and to ensure samples were free of contamination. All samples had absorbance 260/230 ratios of between 1.7 and 2.0, and 260/280 values between 1.8 to 2.1. Each RNA sample (5ul) was used to determine RNA integrity analysis (RIN) Total RNA quality was assessed using the Agilent 4200 TapeStation with RNA ScreenTape® (Agilent Technologies) and quantity with the Invitrogen™ Qubit-iT™ RNA HS Assay Kit (Fisher Scientific) according to the manufacturer’s instructions. Libraries were prepared from 300ng of total RNA with a RIN value > 7. Total RNA was depleted of ribosomal RNA and sequencing libraries prepared with the Illumina®TruSeq Stranded Total RNA Library Prep Gold (Illumina, Inc) kit using TruSeq CD Index Adapters1 (Illumina, Inc). The steps included the depletion of cytoplasmic and mitochondrial rRNA, depleted RNA fragmentation, 1st strand cDNA synthesis, 2nd strand cDNA synthesis, adenylation of 3’ ends, adapter ligation, DNA fragment enrichment by PCR amplification (10-cycles) and validation. The manufacturer’s instructions were followed except for the cleanup after the ribozero depletion step where Ampure®XP beads (Beckman Coulter) and 80% Ethanol were used. The libraries were validated using the Agilent 4200 TapeStation® with high-sensitivity D1000 ScreenTape® (Agilent Technologies) to ascertain the insert size, and the Invitrogen™ Qubit™ dsDNA HS Assay Kit (Fisher Scientific) used to perform the fluorometric quantitation. Following validation, the libraries were normalized to 4 nM, pooled together and clustered on the cBot™2 (Illumina, Inc) following the manufacturer’s recommendations.
characteristics.characteristic: description
characteristics.text: adult
characteristics.characteristic: developmental stage
characteristics.text: wild type genotype
characteristics.characteristic: genotype
characteristics.text: skin punch biopsy
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: skin of body
characteristics.characteristic: organism part
characteristics.text: Mus musculus
characteristics.characteristic: scientific_name
characteristics.text: male
characteristics.characteristic: sex
characteristics.text: C57BL/6
characteristics.characteristic: strain
characteristics.text: R087-H-020
characteristics.characteristic: title