2025-12-05
ENA first public
ERC000011
ENA-CHECKLIST
UCL
INSDC center name
public
INSDC status
ERS25089323
SRA accession
E-MTAB-15282:cortex_912979
Submitter Id
3
month
age
ArrayExpress
broker name
not collected
collection date
house mouse
common name
Protocols: Total RNA was extracted from 3-month mouse brain from four distinct brain regions: cerebellum, cortex, hippocampus and striatum (Supplementary Table S1) using the RNeasy Mini Kit (Qiagen). Total RNA was extracted from 3-month mouse spleen, heart and quadricep muscle (Supplementary Table S1) using the RNeasy Fibrous Tissue Mini Kit (Qiagen). All tissue was homogenised on ice using a TissueRuptor II (QIAGEN) in Buffer RLT with 1% β-mercaptoethanol, following the manufacturer’s instructions for both kits.  The final extracted RNA was eluted in DNase- and RNase-free water. RNA was quantified using a NanoDrop spectrophotometer followed by RNA Integrity Analysis. cDNA synthesis was carried out using Superscript™ IV First-Strand Synthesis System (11904-018; Thermo Fisher Scientific). 2 μg RNA was used as input for annealing of oligo-dT primers. Final clean-up was performed with 1 μL of RNase H. RT-PCR was performed using LongAmp® Taq DNA Polymerase (New England Biolabs). Optimal annealing temperature was determined using a gradient thermocycler. Primers were designed and screened for specificity using NCBI's Primer-BLAST. The primers were positioned to anneal to conserved, non-coding regions flanking the longest common sequence, allowing for the amplification of all Taf1 mRNA variants, from the 5' untranslated region (UTR) (Forward primer sequence: AAGTCACCCGTGTGCGACTGACG) through to the 3' UTR (Reverse primer sequence: AGCTGGGGGGAAAGGTAATAGACC). For each sample, duplicate PCRs were performed in reaction volumes of 50 μL with 0.1 mM of each primer. Thermocycling conditions were an initial denaturation step at 94 °C for 30 seconds, followed by 29 cycles of 94 °C for 30 seconds for denaturation, 61 °C for 30 seconds for    annealing, and 65 °C for 12 minutes for extension. This was followed by a final extension at 65 °C for 10 minutes, and the reactions were held at 4 °C indefinitely. The number of cycles was optimized at 29 to ensure adequate amplification of the target sequences while minimizing non-specific products. Following amplification, the duplicate samples were pooled and the size of the RT-PCR product verified by agarose gel electrophoresis in 1x TAE buffer using SYBR Safe DNA stain for visualisation. The remaining PCR product (~80 μL) was sent to Full Circle Laboratories and purified using 0.5x AMPure XP beads (Beckman Coulter, Fullerton, USA) before proceeding to library construction.
description
adult
developmental stage
wild type genotype
genotype
not collected
geographic location (country and/or sea)
912979
individual
not applicable
isolate
Mus musculus
organism
Mus musculus
organism
cortex
organism part
Mus musculus
scientific_name
female
sex
C57BL/6J
strain
cortex_912979
title