GSM2587312: RongchangPig2_Brain; Sus scrofa; RNA-Seq
Total RNA was extracted using the TRIzol (Life Technologies, Beijing, China) method following manufacturer’s protocol. RNA quality was confirmed using gel electrophoresis and visual spectrophotometry using a Nanodrop spectrophotometer. All samples had a 260/280 nm absorbance ratios between 1.8 and 2.0. Sequencing libraries were constructed according to the manufacturer's standard procedures (Illumina, San Diego, USA). In brief, the poly(A) mRNA was extracted from total RNA using poly-T-attached magnetic beads, further fragmented, and used as templates for cDNA generation. The first-strand was synthesized using reverse transcriptase and random hexamer primers. Second-strand cDNA was synthesized based on the first strand with dNTP (here, dTTP was replaced by dUTP), buffer solution, DNA polymerase I, and RNase H. Double-stranded cDNA was then purified by AMPure XP beads, and the second strands containing U were degraded using the USER enzyme. Purified double-stranded cDNA was end-repaired, an A tail was added, and ligated to paired-end Illumina sequencing adaptors. About 320-bp cDNA fragments were size-selected by AMPure XP beads and amplified using PCR. The high-quality libraries were sequenced on the Illumina HiSeq 2000 platform and 100-bp paired-end reads were obtained. Bases were called using the Illumina CASAVA software.