GSM9045388: PEM-3; Gallus gallus; RNA-Seq
Total RNA was extracted from chicken skeletal muscle tissue using TRIzol reagent (Invitrogen, USA) according to the manufacturer's instructions. Briefly, approximately 100 mg of frozen muscle tissue was homogenized in 1 mL of TRIzol using a tissue grinder under liquid nitrogen. After incubation at room temperature for 5 minutes to permit complete dissociation of nucleoprotein complexes, 200 µL of chloroform was added per 1 mL of TRIzol. The samples were shaken vigorously for 15 seconds and then incubated at room temperature for 2–3 minutes. Phase separation was achieved by centrifugation at 12,000 × g for 15 minutes at 4°C. The upper aqueous phase containing RNA was carefully transferred to a new RNase-free microcentrifuge tube, and an equal volume of isopropanol was added to precipitate RNA. After incubation at room temperature for 10 minutes, the mixture was centrifuged at 12,000 × g for 10 minutes at 4°C. The resulting RNA pellet was washed once with 1 mL of 75% ethanol (prepared with DEPC-treated water), followed by centrifugation at 7,500 × g for 5 minutes at 4°C. After discarding the supernatant, the pellet was air-dried at room temperature for 5–10 minutes and then dissolved in 30–50 µL of RNase-free water.  RNA purity and quantification were evaluated using the NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). RNA integrity was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The libraries were constructed using VAHTS Universal V6 RNA-seq Library Prep Kit according to the manufacturer's instructions. The transcriptome sequencing and analysis were conducted by OE Biotech Co., Ltd. (Shanghai, China).