PTH cDNA library construction, deep sequencing of Tupaia transcriptome, and bioinformatics analysis of Illumina deep sequencing-determined transcriptome Primary Tupaia hepatocytes were isolated as described above. PTH mRNA was purified from 10 μg of total RNA using Oligo-dT magnetic beads. The mRNA was fragmented into small pieces by incubation with divalent cations at 94°C for exactly 5 min. The first strand cDNA was synthesized using random primers and SuperScript II reverse transcriptase (Invitrogen) with fragmented mRNAs. RNA template was then removed by RNase H, and double-stranded cDNA was prepared with DNA polymerase I. cDNA with blunt ends was created by T4 DNA polymerase and Klenow DNA polymerase and an 'A' base was subsequently added to the 3′ end of the blunt phosphorylated DNA fragments by Klenow fragment (3′ to 5′ exo minus). The cDNA was then ligated with adapters and then ran on a 2% agarose gel. The fragments with a size range from 200 ± 25 bp were purified, followed by amplification using the manufacturer's primers. The PCR products were then purified using QIAquick PCR purification Kit (Qiagen), quantified and diluted for cluster generation and deep sequencing. The 72-cycle pair-end sequencing was performed with Sequencing Kits (Version 5) on an Illumina Genome Analyzer IIx (Illumina, San Diego, United States). Illumina CASAVA pipeline v1.8.1 was used for sequence extraction and filtering.