RNA-Seq of Gallus gallus: adult male cecum
Total RNA was extracted from cecum tissue using RNAiso Plus Total RNA extraction reagent (Takara) following the manufacturers instructions, and RNA was detected with a NanoDrop2000 spectrophotometer (Thermo Fisher Scientific, DE, United States). The quantity of total RNA was determined using a Qubit 2.0 fluorimeter (Life Technologies, CA, United States), and the integrity of total RNA was analyzed using a Bioanalyzer 2100 system (Agilent Technologies, CA, United States). After the RNA samples were qualified, the eukaryotic mRNA was enriched by magnetic beads with oligo (dT). Then, cDNA was synthesized using mRNA as template. Second-strand cDNA was synthesized by buffer, dNTPs, DNA polymerase I and RNase H and purified using AMPure XP Beads. The purified double-stranded cDNA was end repaired; poly(A) was added, and the cDNA was ligated to a sequencing connector. After fragment selection and PCR amplification, a sequencing library was obtained.