title: GSM7073781: cow,thermoneutrality, rep1; Bos taurus; RNA-Seq
library_construction_protocol: Total RNA was isolated from each sample using TRIzol reagent (Invitrogen, Carlsbad, CA), Phasemaker Tubes (Invitrogen), and RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions with minor modifications. Specifically, tissue samples were homogenized in TRIzol and lysates were transferred to Phasemaker Tubes with chloroform for 20 min rotation at 4°C. The aqueous phase was then transferred to the gDNA elimination column for 1 min rotation at room temperature. The eluted RNA was washed with cold 70% ethanol, transferred to the RNeasy mini spin column, and washed with buffer RPE and RW1 (RNeasy Mini Kit). Finally, RNAs were eluted in RNAse-free water and concentrations were determined by a NanoDrop 1000 spectrophotometer (Thermo Scientific, Waltham, MA). RNA quality was evaluated on Bioanalyzer 2100 (Agilent) and only samples passed the QC were proceed with the downstream process. Next, mRNA was enriched from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers followed by the second strand cDNA synthesis. The library was ready after a series of subsequent steps, including end pair, A-tailing, adapter ligation, size selection, amplification, and purification. Finally, the library was checked with Qubit and real-time PCR for qualification and evaluated on a bioanalyzer for fragment size distribution. Quantified libraries were pooled and sequenced on Illumina platforms in paired-end mode (2x150bp).