Nanodroptm 1000 spectrometer

To measure antigen specific gene expression 2 Â 10 6 PBMC were stimulated overnight (o/n) with PPD-B (10 mg/ml), Ag85A (5 mg/ml) or media. PBMC were then lysed in lysis buffer containing TCEP (Qiagen) and kept at À80 °C until further use. RNA of PBMC was extracted using the Qiagen RNeasy Minikit according to manufacturer's protocol and RNA quantity was measured using a NanoDrop TM 1000 spectrometer (Thermo Fisher, UK). RNA was transcribed into cDNA using the Invitrogen TM SuperScript TM VILO TM kit (Sigma) and qPCR was performed using TaqMan Ò Assay (Thermo Fisher Scientific). Gene expression is presented as fold change, calculated using the 2 ÀDDCT method [16] and normalised against the geometrical mean of three reference genes (GAPDH, SDHA and YWAHZ).

Blood samples were collected from 632 Chinese Tibetan sheep representing three breeds: Black Tibetan sheep (BT, N = 226), Gaoyuan Tibetan sheep (GT, N = 191), and Oula Tibetan sheep (OT, N = 215). These three groups represent the main Chinese breeds that are reared in the Provinces of Qinghai, Gansu, and Henan, respectively. Their growth traits (body weight, body height, body length, and chest circumference) were recorded at 3 years of age.
Genomic DNA was extracted from sheep blood (jugular vein samples) by the standard phenol-chloroform extraction procedure (He et al., 2012) . DNA quantity and purity (A 260 / A 280 ratio) for each sample was assessed using a Nano-Drop TM 1000 Spectrometer (Thermo Scientific, Waltam, MA, USA).