Chicken preadipocytes differentiation
Primary culture of chicken preadipocytes from abdominal adipose tissue  Chicken preadipocytes from abdominal adipose tissue were cultured according to the method described by (Shang et al. 2014), with some modifications. Abdominal adipose tissue weighing approximately 4 g was collected from three 14-day-old Jinghai Yellow chickens under sterile conditions. Adipose tissue was washed with phosphate-buffered saline (PBS) supplemented with penicillin (100 units/mL) and streptomycin (100 µg/mL). The washed tissue was cut into 1-mm3 sections with a surgical scissors, and digested in 2 mg/mL collagenase type I (Sangon Biotech, Shanghai, China) with shaking for 65 min at 37°. The digested cell suspension was filtered using 200 and 500 mesh screens, and centrifuged at 300 g for 10 min (22°) to separate the stromal-vascular fractions from undigested tissue debris and mature adipocytes. Stromal-vascular cells were plated on a 60 mm culture plate at a density of 1 × 105 cells/mL, and cultured with DMEM/F12 (Dulbecco’s modified Eagle’s medium/Ham’s nutrient mixture F-12) and basic medium (10% [v/v] fetal bovine serum [FBS], 100 units/mL penicillin, and 100 µg/mL streptomycin) in a humidified atmosphere with 5% (v/v) CO2 at 37° until reaching 90% confluence.  Induction of abdominal preadipocytes  After achieving 90% cell confluence, the cells were passaged to 12-well plates and cultured until achieving 90% confluence yet again. The basic medium was then removed and replaced with differentiation medium (0.25 µM dexamethasone [Takara], 10 µg/mL insulin [Takara], and 0.5 mM 3-isobutyl-1-methylxanthine [IBMX, Takara] ) for 48 h. The differentiation medium was replaced with maintenance medium (10 µg/mL insulin [Takara]) and incubated for 48 h. Cells were collected after being induced for 0, 48, 96, and 144 h (0, 2, 4, and 6 days). Each interval included three biological replicates (n = 3).  RNA extraction, library construction, and sequencing  A total of 12 cell samples were successfully collected. Total RNA was extracted using Trizol reagent (Invitrogen, Hong Kong, China). The purity, concentration, and integrity of total RNA were checked using the Nanodrop spectrophotometer, Qubit 2.0 fluorometer, and Aglient 2100 Bioanalyzer, respectively. All RNA samples had an RNA integrity number (RIN) value greater than 8.0, an optical density (OD) 260/280 ratio greater than 1.9, and were selected for library construction and deep sequencing. The rRNA was removed and mRNA was enriched using magnetic beads with oligo (dT) primer and then randomly fragmented using Fragmentation buffer. The mRNA was used as a template to synthesize the first-strand cDNA, using 1st Strand Enzyme Mix (Vazyme, Nanjing, China). The second-strand cDNA was synthesized using 2nd Strand Marking Buffer and 2nd Strand/End Repair Enzyme Mix (Vazyme). The products were purified by VAHTS™ DNA Clean Beads and the end of the double strand was then repaired and A-tailed. An adapter was joined to A-tailed products using ligation mix. Suitably sized fragments were selected using VAHTS™ DNA Clean Beads to construct the cDNA library by PCR. The RNA sequencing was performed using Illumina XTen (Vazyme Biotech Co., Ltd).
Yangzhou University
Chicken abdominal preadipocytes