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[1], with some modifications. Abdominal adipose tissue of was collected from 14-day-old Jinghai yellow chicken under sterile condiction. Adipose tissue was washed by PBS supplemented with penicillin (100 units/ml) and streptomycin (100 μg/ml). The washed tissue was cut to 1㎜3 by surgical scissors and then digested using 2 mg/ml collagenase type I (Sangon Biotech) with shaking for 65 min at 37 ◦C. The digested cell suspension was filtrated using 200 and 500 mesh screens and centrifuged at 300g for 10min (22 ◦C) to separate the stromal-vascular fractions from undigested tissue debris and mature adipocytes. Stromal-vascular cells were plated to 50mm culture palte 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) basic medium (10% (v/v) FBS, 100 units/ml penicillin and 100 μg/ml streptomycin) in a humidified atmosphere with 5% (v/v) CO2 at 37 ◦C until reaching 90％confluence.  Induction of abdominal preadipocytes  Following 90％cell confluence, the basic medium was removed and replaced with differentiation medium (0.25 μM dexamethasone (Takara), 10 μg/ml insulin (Takara) and 0.5 mM IBMX (Takara) for 48 hours. The differentiation medium was replaced with maintenance medium (10 μg/ml insulin (Takara)) and incubated for 48 hours.  The detailed procedure for induction of abdominal preadipocytes was described in Fig.1. Cells were collected after induced for 0h, 48h, 96h and 144h. Each point included 3 biological replicates (n=3).     Total RNA was extracted using Trizol reagent (Invitrogen). The integrity, concentration and purity of total RNA were checked via Nanodrop, Qubit 2.0 and Aglient 2100. RNA samples with a RIN value greater than 8.0 and an OD 260/280 ratio greater than 1.9 were selected for deep sequencing.The rRNA was removed and mRNA was enriched using magnetic bead with Oligo (dT) and then randomly fragmented by Fragmentation Buffer. The mRNA was used as template to synthesize the first-strand cDNA using 1st Strand Enzyme Mix (Enzyme). The second-strand cDNA was synthesized using 2nd Strand Marking Buffer and 2nd Strand/End Repair Enzyme Mix (Enzyme). The products were purified by VAHTSTM DNA Clean Beads and then the end of double strand was repaired and A-tailed. An adapter was jointed to A-tailed products using ligation mix. Suitable sized fragments were selected using VAHTSTM DNA Clean Beads to construct the cDNA library by PCR. The RNA sequencing was performed using Illumina X Ten.