Illumina HiSeq 2500 paired end sequencing; Transcriptome profiling of Bma1 knock out mouse by RNASeq
Transcriptome profiling of Bma1 knock out mouse by RNASeq
In this experiment two mouse lines (WT and Bmal1 knockout) were studied for circadian gene expression at four time points in a 24 hour period. All animal studies were performed in accordance with the 1986 UK Home Office Animal Procedures Act. Approval was provided by the local ethics committee. Mice were maintained at 20-22oC, with standard rodent chow available ad libitum and under 12:12 hr light dark schedule. Bmal1flox/flox mice were crossed onto a PER2::luc background. The PER2::luc mice carry the firefly luciferase gene fused in-frame with the 3’ end of the Per2 gene, creating a fusion protein reporter. Bmal1flox/flox - PER2::luc mice were subsequently crossed with Col2a1cre mice expressing cre recombinase under the control of the Col2a1 promoter20 to generate cartilage specific Bmal1 knock-out. All mice were bred in-house at the University of Manchester. Hip cartilage was obtained from 8-10 weeks old mice by dislocating the joint and peeling off the cartilage from the head of the femur using a scalpel blade. The tissue was immediately snap frozen in liquid nitrogen, and then stored at -80oC until mRNA extraction. Tissue was homogenised using a Mikro-Dismembrator S (Satorius Stedim Biotech) with the barrel and ball of the dismembrator pre-cooled in liquid nitrogen. mRNA was extracted using RNeasy micro kit (Qiagen) according to the manufacturer’s protocol. Illumina TruSeq Stranded mRNA Library Prep Kit
Experimental Factor: time
09:00 PM
Experimental Factor: genotype
wild type genotype
LIBRARY_STRAND
first strand