GSM5600277: 1702-0235-L; Sus scrofa; RNA-Seq
Total DNA from minipig blood sample was subjected to whole genome sequencing using the Illumina Paired-end sequencing on an Illumina platform. Total RNA from Liver and Backfat were prepared using TRIzol (Invitrogen) and a RNeasy RNA Purification Kit with DNase treatment (Qiagen), according to the instruction manual.  One microliter of cleaned total RNA was consumed to check RNA quality using BioAnalyzer 2011 with an RNA chip (RIN > 7 and 28S:18S ratio > 1.0). Messenger RNA sequencing libraries were generated using an mRNA Seq Sample Prep Kit following the manufacturer's manual (Illumina). 5 ug of poly(A) mRNA was isolated from the total RNA using oligo-dT beads. The mRNA was then fragmented and randomly primed for reverse transcription, followed by the second-strand synthesis to create double-stranded cDNA fragments. The double-stranded short cDNA fragments were purified with a QiaQuick PCR extraction kit (Qiagen) and resuspended in an EB buffer. Each end of cDNA was repaired with a combination of fill-in reactions and blunted by exonuclease. The addition of A-base was applied to the blunt end DNA followed by sequencing adaptor ligation. The required fragments were purified by agarose gel electrophoresis and a QIAquick Gel Extraction Kit (Qiagen), and enriched by PCR amplification. Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit.