GSM2101067: N; Bos taurus; RNA-Seq
At least 4 ml blood was used for RNA extraction from each sample. First, buffy coat of blood samples was isolated by centrifugation at 2000 ×g for 10 min at 4 0C, then white blood cells (WBC) were washed with red blood cell (RBC) lysis buffer (1X) twice and centrifuged at 2000 ×g for 10 min at 4 0C to obtain white pellet of WBC. Total RNA was extracted using Trizol reagent method. Total RNA was digested by DNaseІ (NEB) and purified by oligo-dT beads (Dyna beads mRNA purification kit, Invitrogen, USA), then poly (A)-containing mRNA were fragmented into 130 bp with first strand buffer. First-strand cDNA was synthesized using N6 primer, first strand master mix, and super script II reverse transcription (Invitrogen, USA). Reaction condition: 25°C for 10 min; 42°C for 40 min; 70°C for 15 min. The second strand master mix was added to synthesize the second-strand cDNA (16°C for 1h). The cDNA was Purified using Agencourt® AMPure® XP PCR purification kit (Agencourt, USA). The final library was quantitated in two ways for validation: the average of molecule length was determined using the Agilent 2100 Bioanalyzer instrument (Agilent DNA 1000 Reagents), and quantification of the library was performed by real-time quantitative PCR (qPCR) using TaqMan Probes. The qualified libraries were amplified on cBot to generate the cluster on the flow cell (TruSeq PE cluster kit V3–cBot–HS, Illumina). The amplified flow cell was sequenced, pair ended, on the HiSeq 2000 device (TruSeq SBS KIT-HS V3, Illumina) with read length of 100 base pair.
GEO
GSM2101067