title: Illumina Genome Analyzer IIx paired end sequencing; RNA-seq of liver samples from adult Ryukyu mice (Mus caroli)
design: RNA-seq of liver samples from adult Ryukyu mice (Mus caroli)
library_construction_protocol: Mice were housed in the Cambridge Research Institute Biological Resource Unit with a 12-h light/dark cycle, and were provided with chow food from Lab Diet plus water ad libitum. Mice were sacrificed by cervical dislocation at 4–6 mo of age, between the hours of 9:30am and 11:30am. Each mouse was perfused with PBS and the liver removed. A sample of 50–100 mg was taken from a single liver lobe and frozen in liquid nitrogen for storage at −80°C until use. Gonclaves et al. 2012. Extensive compensatory cis-trans regulation in the evoluition of mouse gene expression. Genome research. 22:2376-2384 For each liver sample, total RNA was extracted using Qiazol (Qiagen) as per manufacturer's instructions and DNase treated with DNA-free (Ambion). Polyadenylated mRNA was enriched from the total RNA using the PolyATract mRNA isolation system (Promega). Directional double-stranded cDNA was generated according to the method of Parkhomchuk et al. (2009), using the Superscript Double-Stranded cDNA Synthesis kit (Invitrogen), with uracil substituted for thymine in the second strand; 250 ng of double-stranded cDNA was fragmented by sonication and a sequencing library prepared for the Illumina platform using the Paired End Oligo Only kit (Illumina) according to the manufacturer's instructions. Strand specificity was then introduced by digestion of the second strand of cDNA using uracil-N-glycosylase. Each library was PCR-amplified using Illumina's PE primers. Size selection was performed by 2% agarose gel electrophoresis and 200–300-bp fragments were extracted using a Minelute gel purification kit (Qiagen).
attributes.tag: Experimental Factor: individual
attributes.value: 0UH99