cortex_912985
Mus musculus
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.
ENA first public
2025-12-05
INSDC center name
UCL
INSDC status
public
Submitter Id
E-MTAB-15282:cortex_912985
age
3
broker name
ArrayExpress
collection date
not collected
common name
house mouse
developmental stage
adult
genotype
wild type genotype
geographic location (country and/or sea)
not collected
individual
912985
isolate
not applicable
organism part
cortex
sample name
E-MTAB-15282:cortex_912985
scientific_name
Mus musculus
sex
female
strain
C57BL/6J