GSM7968036: MAC-T cells, heat stress differentiated and recovered 6hr, rep3; Bos taurus; RNA-Seq
Total RNA was isolated from cell pellets with TRIzol reagent (Invitrogen), Phasemaker tubes (Invitrogen), and RNeasy Mini Kit (Qiagen, Germany) according to the manufacturer's instructions with minor modifications. Cell pellets were homogenized in 1 mL TRIzol reagent using a pellet mixer (VWR International, PA) for 30s, and lysates were transferred to Phasemaker tubes with chloroform for 20 min centrifugation at 4°C. The aqueous phase was then transferred to the gDNA elimination column for 1 min centrifugation at room temperature. The eluted RNA was washed with cold 50% 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. Concentrations of RNA samples were determined by a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific), and samples with 260/280 and 260/230 ratios around 2 were considered as pure RNA. Qualities of RNA samples were determined by an Agilent 2100 Bioanalyzer (Agilent Technology, CA), and all samples has high RNA integrity number (RIN) larger than 9. Two technical replicates of each group with high concentration and quality were pooled for RNA sequencing. The quality control, library construction, and RNA sequencing of RNA samples were conducted by Novogene Inc. (Sacramento, CA). Briefly, messenger RNA (mRNA) was purified from total RNA using poly-T selection. mRNA was then fragmented and reverse transcribed. Following end-repairing and A-tailing, cDNA was ligated with adapters and selected based on fragment sizes. Finally, PCR amplification was performed, and quantified libraries were pooled and sequenced on Illumina platforms to generate paired-end raw reads. From the raw sequencing data, adaptors were removed, and quality control was conducted using fastp (v0.23.2) (Chen et al., 2018). Reference genome of bus taurus (ARS-UCD 1.3) was downloaded from the UCSC database, and alignment was conducted using STAR (v2.7.9a) (Dobin et al., 2013).