title: R087-H-018
organism: Mus musculus
description: 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.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2023-06-21
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2023-06-21
attributes.tag: External Id
attributes.value: SAMEA112890773
attributes.tag: INSDC center alias
attributes.value: CARDIFF UNIVERSITY
attributes.tag: INSDC center name
attributes.value: CARDIFF UNIVERSITY
attributes.tag: INSDC first public
attributes.value: 2023-06-21T00:18:24Z
attributes.tag: INSDC last update
attributes.value: 2023-06-21T00:18:24Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-12845:R087-H-018
attributes.tag: age
attributes.value: 8 to 12
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: house mouse
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: injury
attributes.value: skin punch biopsy
attributes.tag: isolate
attributes.value: not applicable
attributes.tag: organism part
attributes.value: skin of body
attributes.tag: sample name
attributes.value: E-MTAB-12845:R087-H-018
attributes.tag: scientific_name
attributes.value: Mus musculus
attributes.tag: sex
attributes.value: male
attributes.tag: strain
attributes.value: C57BL/6