RNA extraction and RNA-seq

The CAM tissues of embryos at day 11 of incubation were used in RNA-Seq. Total RNA was extracted using RNA pure Tissue Kit (Tiangen Biochemical Technology Beijing Co., Ltd) according to the corresponding manufacturer’s protocol. Six RNA-Seq libraries were constructed, including three Tibetan chickens (TC1, TC2, TC3) and three Chahua chickens (CH1, CH2, CH3) incubated in hypoxic conditions. Each sample was mixed from three individuals in the same breed. The pooled RNA samples were purified with a RNeasy Micro Kit (Cat. #74004; QIAGEN, Venlo, Netherlands) for cDNA library preparation (approximately 3 μg of total RNA). Poly(A) mRNA isolation, first-and second-strand cDNA synthesis, and fragment, connecting adapter, and cDNA library preparation were performed sequentially with the TruSeq RNA Sample Prep Kit (Cat. #RS-122-2002; Illumina, San Diego, CA, USA) following the manufacturer’s protocol⁵¹. All libraries were sequenced using the Illumina HiSeq 4,000 platform for paired-end 150-bp sequencing.