RNA-Seq  of Gallus_gallus
A total  amount of 3g RNA per sample was used as input material for the RNA sample  preparations. Firstly, ribosomal RNA was removed by Epicentre Ribo-zero rRNA  Removal Kit (Epicentre,USA), and rRNA free residue was cleaned up by ethanol  precipitation. Subsequently, sequencing libraries were generated using the  rRNAdepleted RNA by NEBNext Ultra Directional RNA Library Prep Kit for  Illumina (NEB,USA) following manufacturer's recommendations. Briefly,  fragmentation was carried out using divalent cations under elevated  temperature in NEBNext First Strand Synthesis Reaction Buffer(5X). First  strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse  Transcriptase(RNaseH-). Second strand cDNA synthesis was subsequently  performed using DNA Polymerase I and RNase H. In the reaction buffer, dNTPs  with dTTP were replaced by dUTP. Remaining overhangs were converted into  blunt ends via exonuclease/polymerase activities. After adenylation of 3  ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were  ligated to prepare for hybridization. In order to select cDNA fragments of  preferentially 150-200 bp in length, the library fragments were purified with  AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 l USER Enzyme (NEB,  USA) was used with size-selected, adaptor-ligated cDNA at 37C for 15 min  followed by 5 min at 95C before PCR. Then PCR was performed with Phusion  High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At  last, products were purified (AMPure XP system) and library quality was  assessed on the Agilent Bioanalyzer 2100 system.